Yun, Bing-Ling; Guan, Xiao-Lu; Liu, Yong-Zhen; Zhang, Yao; Wang, Yong-Qiang; Qi, Xiao-Le; Cui, Hong-Yu; Liu, Chang-Jun; Zhang, Yan-Ping; Gao, Hong-Lei; Gao, Li; Li, Kai; Gao, Yu-Long; Wang, Xiao-Mei
2016-07-08
Avian metapneumovirus (aMPV) fusion (F) protein mediates virus-cell membrane fusion to initiate viral infection, which requires F protein binding to its receptor(s) on the host cell surface. However, the receptor(s) for aMPV F protein is still not identified. All known subtype B aMPV (aMPV/B) F proteins contain a conserved Arg-Asp-Asp (RDD) motif, suggesting that the aMPV/B F protein may mediate membrane fusion via the binding of RDD to integrin. When blocked with integrin-specific peptides, aMPV/B F protein fusogenicity and viral replication were significantly reduced. Specifically we identified integrin αv and/or β1-mediated F protein fusogenicity and viral replication using antibody blocking, small interfering RNAs (siRNAs) knockdown, and overexpression. Additionally, overexpression of integrin αv and β1 in aMPV/B non-permissive cells conferred aMPV/B F protein binding and aMPV/B infection. When RDD was altered to RAE (Arg-Ala-Glu), aMPV/B F protein binding and fusogenic activity were profoundly impaired. These results suggest that integrin αvβ1 is a functional receptor for aMPV/B F protein-mediated membrane fusion and virus infection, which will provide new insights on the fusogenic mechanism and pathogenesis of aMPV. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
Yun, Bing-Ling; Guan, Xiao-Lu; Liu, Yong-Zhen; Zhang, Yao; Wang, Yong-Qiang; Qi, Xiao-Le; Cui, Hong-Yu; Liu, Chang-Jun; Zhang, Yan-Ping; Gao, Hong-Lei; Gao, Li; Li, Kai; Gao, Yu-Long; Wang, Xiao-Mei
2016-01-01
Avian metapneumovirus (aMPV) fusion (F) protein mediates virus-cell membrane fusion to initiate viral infection, which requires F protein binding to its receptor(s) on the host cell surface. However, the receptor(s) for aMPV F protein is still not identified. All known subtype B aMPV (aMPV/B) F proteins contain a conserved Arg-Asp-Asp (RDD) motif, suggesting that the aMPV/B F protein may mediate membrane fusion via the binding of RDD to integrin. When blocked with integrin-specific peptides, aMPV/B F protein fusogenicity and viral replication were significantly reduced. Specifically we identified integrin αv and/or β1-mediated F protein fusogenicity and viral replication using antibody blocking, small interfering RNAs (siRNAs) knockdown, and overexpression. Additionally, overexpression of integrin αv and β1 in aMPV/B non-permissive cells conferred aMPV/B F protein binding and aMPV/B infection. When RDD was altered to RAE (Arg-Ala-Glu), aMPV/B F protein binding and fusogenic activity were profoundly impaired. These results suggest that integrin αvβ1 is a functional receptor for aMPV/B F protein-mediated membrane fusion and virus infection, which will provide new insights on the fusogenic mechanism and pathogenesis of aMPV. PMID:27226547
Discoidin domain receptor 1 is activated independently of beta(1) integrin
DEFF Research Database (Denmark)
Vogel, W; Brakebusch, C; Fässler, R
2000-01-01
independent of the epidermal growth factor (EGF) receptor. In cells that endogenously express both DDR1 and the EGF receptor, stimulation with EGF does not induce DDR activation. Third, we detected full DDR1 activation after collagen stimulation in cells that have been treated with blocking antibodies...... for alpha(2)beta(1) integrin or in cells with a targeted deletion of the beta(1) integrin gene. Finally, we show that overexpression of dominant negative DDR1 in the myoblast cell line C2C12 blocks cellular differentiation and the formation of myofibers....
Mechanisms of integrin-vascular endothelial growth factor receptor cross-activation in angiogenesis.
Mahabeleshwar, Ganapati H; Feng, Weiyi; Reddy, Kumar; Plow, Edward F; Byzova, Tatiana V
2007-09-14
The functional responses of endothelial cells are dependent on signaling from peptide growth factors and the cellular adhesion receptors, integrins. These include cell adhesion, migration, and proliferation, which, in turn, are essential for more complex processes such as formation of the endothelial tube network during angiogenesis. This study identifies the molecular requirements for the cross-activation between beta3 integrin and tyrosine kinase receptor 2 for vascular endothelial growth factor (VEGF) receptor (VEGFR-2) on endothelium. The relationship between VEGFR-2 and beta3 integrin appears to be synergistic, because VEGFR-2 activation induces beta3 integrin tyrosine phosphorylation, which, in turn, is crucial for VEGF-induced tyrosine phosphorylation of VEGFR-2. We demonstrate here that adhesion- and growth factor-induced beta3 integrin tyrosine phosphorylation are directly mediated by c-Src. VEGF-stimulated recruitment and activation of c-Src and subsequent beta3 integrin tyrosine phosphorylation are critical for interaction between VEGFR-2 and beta3 integrin. Moreover, c-Src mediates growth factor-induced beta3 integrin activation, ligand binding, beta3 integrin-dependent cell adhesion, directional migration of endothelial cells, and initiation of angiogenic programming in endothelial cells. Thus, the present study determines the molecular mechanisms and consequences of the synergism between 2 cell surface receptor systems, growth factor receptor and integrins, and opens new avenues for the development of pro- and antiangiogenic strategies.
PTP1B triggers integrin-mediated repression of myosin activity and modulates cell contractility
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Ana E. González Wusener
2016-01-01
Full Text Available Cell contractility and migration by integrins depends on precise regulation of protein tyrosine kinase and Rho-family GTPase activities in specific spatiotemporal patterns. Here we show that protein tyrosine phosphatase PTP1B cooperates with β3 integrin to activate the Src/FAK signalling pathway which represses RhoA-myosin-dependent contractility. Using PTP1B null (KO cells and PTP1B reconstituted (WT cells, we determined that some early steps following cell adhesion to fibronectin and vitronectin occurred robustly in WT cells, including aggregation of β3 integrins and adaptor proteins, and activation of Src/FAK-dependent signalling at small puncta in a lamellipodium. However, these events were significantly impaired in KO cells. We established that cytoskeletal strain and cell contractility was highly enhanced at the periphery of KO cells compared to WT cells. Inhibition of the Src/FAK signalling pathway or expression of constitutive active RhoA in WT cells induced a KO cell phenotype. Conversely, expression of constitutive active Src or myosin inhibition in KO cells restored the WT phenotype. We propose that this novel function of PTP1B stimulates permissive conditions for adhesion and lamellipodium assembly at the protruding edge during cell spreading and migration.
PTP1B triggers integrin-mediated repression of myosin activity and modulates cell contractility
González Wusener, Ana E.; González, Ángela; Nakamura, Fumihiko; Arregui, Carlos O.
2016-01-01
ABSTRACT Cell contractility and migration by integrins depends on precise regulation of protein tyrosine kinase and Rho-family GTPase activities in specific spatiotemporal patterns. Here we show that protein tyrosine phosphatase PTP1B cooperates with β3 integrin to activate the Src/FAK signalling pathway which represses RhoA-myosin-dependent contractility. Using PTP1B null (KO) cells and PTP1B reconstituted (WT) cells, we determined that some early steps following cell adhesion to fibronectin and vitronectin occurred robustly in WT cells, including aggregation of β3 integrins and adaptor proteins, and activation of Src/FAK-dependent signalling at small puncta in a lamellipodium. However, these events were significantly impaired in KO cells. We established that cytoskeletal strain and cell contractility was highly enhanced at the periphery of KO cells compared to WT cells. Inhibition of the Src/FAK signalling pathway or expression of constitutive active RhoA in WT cells induced a KO cell phenotype. Conversely, expression of constitutive active Src or myosin inhibition in KO cells restored the WT phenotype. We propose that this novel function of PTP1B stimulates permissive conditions for adhesion and lamellipodium assembly at the protruding edge during cell spreading and migration. PMID:26700725
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Michael C Schmid
Full Text Available Tumor inflammation, the recruitment of myeloid lineage cells into the tumor microenvironment, promotes angiogenesis, immunosuppression and metastasis. CD11b+Gr1lo monocytic lineage cells and CD11b+Gr1hi granulocytic lineage cells are recruited from the circulation by tumor-derived chemoattractants, which stimulate PI3-kinase γ (PI3Kγ-mediated integrin α4 activation and extravasation. We show here that PI3Kγ activates PLCγ, leading to RasGrp/CalDAG-GEF-I&II mediated, Rap1a-dependent activation of integrin α4β1, extravasation of monocytes and granulocytes, and inflammation-associated tumor progression. Genetic depletion of PLCγ, CalDAG-GEFI or II, Rap1a, or the Rap1 effector RIAM was sufficient to prevent integrin α4 activation by chemoattractants or activated PI3Kγ (p110γCAAX, while activated Rap (RapV12 promoted constitutive integrin activation and cell adhesion that could only be blocked by inhibition of RIAM or integrin α4β1. Similar to blockade of PI3Kγ or integrin α4β1, blockade of Rap1a suppressed both the recruitment of monocytes and granulocytes to tumors and tumor progression. These results demonstrate critical roles for a PI3Kγ-Rap1a-dependent pathway in integrin activation during tumor inflammation and suggest novel avenues for cancer therapy.
SR-B1 Is a Silica Receptor that Mediates Canonical Inflammasome Activation
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Misato Tsugita
2017-01-01
Full Text Available The inhalation of silica dust is associated with fibrosis and lung cancer, which are triggered by macrophage inflammatory responses; however, how macrophages recognize silica remains largely unknown. Here, we identify by functional expression cloning the class B scavenger receptor SR-B1 as a silica receptor. Through an extracellular α-helix, both mouse and human SR-B1 specifically recognized amorphous and crystalline silica, but not titanium dioxide nanoparticles, latex nanoparticles, or monosodium urate crystals, although all particles exhibited negative surface potentials. Genetic deletion of SR-B1 and masking of SR-B1 by monoclonal antibodies showed that SR-B1-mediated recognition of silica is associated with caspase-1-mediated inflammatory responses in mouse macrophages and human peripheral blood monocytes. Furthermore, SR-B1 was involved in silica-induced pulmonary inflammation in mice. These results indicate that SR-B1 is a silica receptor associated with canonical inflammasome activation.
Diverse roles of integrin receptors in articular cartilage.
Shakibaei, M; Csaki, C; Mobasheri, A
2008-01-01
Integrins are heterodimeric integral membrane proteins made up of alpha and beta subunits. At least eighteen alpha and eight beta subunit genes have been described in mammals. Integrin family members are plasma membrane receptors involved in cell adhesion and active as intra- and extracellular signalling molecules in a variety of processes including embryogenesis, hemostasis, tissue repair, immune response and metastatic spread of tumour cells. Integrin beta 1 (beta1-integrin), the protein encoded by the ITGB1 gene (also known as CD29 and VLAB), is a multi-functional protein involved in cell-matrix adhesion, cell signalling, cellular defense, cell adhesion, protein binding, protein heterodimerisation and receptor-mediated activity. It is highly expressed in the human body (17.4 times higher than the average gene in the last updated revision of the human genome). The extracellular matrix (ECM) of articular cartilage is a unique environment. Interactions between chondrocytes and the ECM regulate many biological processes important to homeostasis and repair of articular cartilage, including cell attachment, growth, differentiation and survival. The beta1-integrin family of cell surface receptors appears to play a major role in mediating cell-matrix interactions that are important in regulating these fundamental processes. Chondrocyte mechanoreceptors have been proposed to incorporate beta1-integrins and mechanosensitive ion channels which link with key ECM, cytoskeletal and signalling proteins to maintain the chondrocyte phenotype, prevent chondrocyte apoptosis and regulate chondrocyte-specific gene expression. This review focuses on the expression and function of beta1-integrins in articular chondrocytes, its role in the unique biology of these cells and its distribution in cartilage.
Human Parechovirus 1 Infection Occurs via αVβ1 Integrin.
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Pirjo Merilahti
Full Text Available Human parechovirus 1 (HPeV-1 (family Picornaviridae is a global cause of pediatric respiratory and CNS infections for which there is no treatment. Although biochemical and in vitro studies have suggested that HPeV-1 binds to αVβ1, αVβ3 and αVβ6 integrin receptor(s, the actual cellular receptors required for infectious entry of HPeV-1 remain unknown. In this paper we analyzed the expression profiles of αVβ1, αVβ3, αVβ6 and α5β1 in susceptible cell lines (A549, HeLa and SW480 to identify which integrin receptors support HPeV-1 internalization and/or replication cycle. We demonstrate by antibody blocking assay, immunofluorescence microscopy and RT-qPCR that HPeV-1 internalizes and replicates in cell lines that express αVβ1 integrin but not αVβ3 or αVβ6 integrins. To further study the role of β1 integrin, we used a mouse cell line, GE11-KO, which is deficient in β1 expression, and its derivate GE11-β1 in which human integrin β1 subunit is overexpressed. HPeV-1 (Harris strain and three clinical HPeV-1 isolates did not internalize into GE11-KO whereas GE11-β1 supported the internalization process. An integrin β1-activating antibody, TS2/16, enhanced HPeV-1 infectivity, but infection occurred in the absence of visible receptor clustering. HPeV-1 also co-localized with β1 integrin on the cell surface, and HPeV-1 and β1 integrin co-endocytosed into the cells. In conclusion, our results demonstrate that in some cell lines the cellular entry of HPeV-1 is primarily mediated by the active form of αVβ1 integrin without visible receptor clustering.
Melchior, Aurélie; Denys, Agnès; Deligny, Audrey; Mazurier, Joël; Allain, Fabrice
2008-02-01
Initially identified as a cyclosporin-A binding protein, cyclophilin B (CyPB) is an inflammatory mediator that induces adhesion of T lymphocytes to fibronectin, by a mechanism dependent on CD147 and alpha 4 beta 1 integrins. Recent findings have suggested that another cell membrane protein, CD98, may cooperate with CD147 to regulate beta1 integrin functions. Based on these functional relationships, we examined the contribution of CD98 in the pro-adhesive activity of CyPB, by utilizing the responsive promonocyte cell line THP-1. We demonstrated that cross-linking CD98 with CD98-AHN-18 antibody mimicked the responses induced by CyPB, i.e. homotypic aggregation, integrin-mediated adhesion to fibronectin and activation of p44/42 MAPK. Consistent with previous data, immunoprecipitation confirmed the existence of a heterocomplex wherein CD147, CD98 and beta1 integrins were associated. We then demonstrated that CyPB-induced cell adhesion and p44/42 MAPK activation were dependent on the participation of phosphoinositide 3-kinase and subsequent activation of protein kinase C-delta. Finally, silencing the expression of CD98 by RNA interference potently reduced CyPB-induced cell responses, thus confirming the role of CD98 in the pro-adhesive activity of CyPB. Altogether, our results support a model whereby CyPB induces integrin-mediated adhesion via interaction with a multimolecular unit formed by the association between CD147, CD98 and beta1 integrins.
Arregui, Carlos O.; Balsamo, Janne; Lilien, Jack
1998-01-01
To investigate the role of nonreceptor protein tyrosine phosphatase 1B (PTP1B) in β1-integrin– mediated adhesion and signaling, we transfected mouse L cells with normal and catalytically inactive forms of the phosphatase. Parental cells and cells expressing the wild-type or mutant PTP1B were assayed for (a) adhesion, (b) spreading, (c) presence of focal adhesions and stress fibers, and (d) tyrosine phosphorylation. Parental cells and cells expressing wild-type PTP1B show similar morphology, are able to attach and spread on fibronectin, and form focal adhesions and stress fibers. In contrast, cells expressing the inactive PTP1B have a spindle-shaped morphology, reduced adhesion and spreading on fibronectin, and almost a complete absence of focal adhesions and stress fibers. Attachment to fibronectin induces tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin in parental cells and cells transfected with the wild-type PTP1B, while in cells transfected with the mutant PTP1B, such induction is not observed. Additionally, in cells expressing the mutant PTP1B, tyrosine phosphorylation of Src is enhanced and activity is reduced. Lysophosphatidic acid temporarily reverses the effects of the mutant PTP1B, suggesting the existence of a signaling pathway triggering focal adhesion assembly that bypasses the need for active PTP1B. PTP1B coimmunoprecipitates with β1-integrin from nonionic detergent extracts and colocalizes with vinculin and the ends of actin stress fibers in focal adhesions. Our data suggest that PTP1B is a critical regulatory component of integrin signaling pathways, which is essential for adhesion, spreading, and formation of focal adhesions. PMID:9813103
Directory of Open Access Journals (Sweden)
Barja-Fidalgo C.
2005-01-01
Full Text Available Extracellular matrix proteins and cell adhesion receptors (integrins play essential roles in the regulation of cell adhesion and migration. Interactions of integrins with the extracellular matrix proteins lead to phosphorylation of several intracellular proteins such as focal adhesion kinase, activating different signaling pathways responsible for the regulation of a variety of cell functions, including cytoskeleton mobilization. Once leukocytes are guided to sites of infection, inflammation, or antigen presentation, integrins can participate in the initiation, maintenance, or termination of the immune and inflammatory responses. The modulation of neutrophil activation through integrin-mediated pathways is important in the homeostatic control of the resolution of inflammatory states. In addition, during recirculation, T lymphocyte movement through distinct microenvironments is mediated by integrins, which are critical for cell cycle, differentiation and gene expression. Disintegrins are a family of low-molecular weight, cysteine-rich peptides first identified in snake venom, usually containing an RGD (Arg-Gly-Asp motif, which confers the ability to selectively bind to integrins, inhibiting integrin-related functions in different cell systems. In this review we show that, depending on the cell type and the microenvironment, disintegrins are able to antagonize the effects of integrins or to act agonistically by activating integrin-mediated signaling. Disintegrins have proven useful as tools to improve the understanding of the molecular events regulated by integrin signaling in leukocytes and prototypes in order to design therapies able to interfere with integrin-mediated effects.
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Alexandra Canonici
Full Text Available Intestinal epithelial cell damage is frequently seen in the mucosal lesions of inflammatory bowel diseases such as ulcerative colitis or Crohn's disease. Complete remission of these diseases requires both the cessation of inflammation and the migration of enterocytes to repair the damaged epithelium. Lyophilized Saccharomyces boulardii (Sb, Biocodex is a nonpathogenic yeast widely used as a therapeutic agent for the treatment and prevention of diarrhea and other gastrointestinal disorders. In this study, we determined whether Sb could accelerate enterocyte migration. Cell migration was determined in Sb force-fed C57BL6J mice and in an in vitro wound model. The impact on α2β1 integrin activity was assessed using adhesion assays and the analysis of α2β1 mediated signaling pathways both in vitro and in vivo. We demonstrated that Sb secretes compounds that enhance the migration of enterocytes independently of cell proliferation. This enhanced migration was associated with the ability of Sb to favor cell-extracellular matrix interaction. Indeed, the yeast activates α2β1 integrin collagen receptors. This leads to an increase in tyrosine phosphorylation of cytoplasmic molecules, including focal adhesion kinase and paxillin, involved in the integrin signaling pathway. These changes are associated with the reorganization of focal adhesion structures. In conclusion Sb secretes motogenic factors that enhance cell restitution through the dynamic regulation of α2β1 integrin activity. This could be of major importance in the development of novel therapies targeting diseases characterized by severe mucosal injury, such as inflammatory and infectious bowel diseases.
Bai, Ming; Grieshaber-Bouyer, Ricardo; Wang, Junxia; Schmider, Angela B; Wilson, Zachary S; Zeng, Liling; Halyabar, Olha; Godin, Matthew D; Nguyen, Hung N; Levescot, Anaïs; Cunin, Pierre; Lefort, Craig T; Soberman, Roy J; Nigrovic, Peter A
2017-11-09
CD177 is a glycosylphosphatidylinositol (GPI)-anchored protein expressed by a variable proportion of human neutrophils that mediates surface expression of the antineutrophil cytoplasmic antibody antigen proteinase 3. CD177 associates with β2 integrins and recognizes platelet endothelial cell adhesion molecule 1 (PECAM-1), suggesting a role in neutrophil migration. However, CD177 pos neutrophils exhibit no clear migratory advantage in vivo, despite interruption of in vitro transendothelial migration by CD177 ligation. We sought to understand this paradox. Using a PECAM-1-independent transwell system, we found that CD177 pos and CD177 neg neutrophils migrated comparably. CD177 ligation selectively impaired migration of CD177 pos neutrophils, an effect mediated through immobilization and cellular spreading on the transwell membrane. Correspondingly, CD177 ligation enhanced its interaction with β2 integrins, as revealed by fluorescence lifetime imaging microscopy, leading to integrin-mediated phosphorylation of Src and extracellular signal-regulated kinase (ERK). CD177-driven cell activation enhanced surface β2 integrin expression and affinity, impaired internalization of integrin attachments, and resulted in ERK-mediated attenuation of chemokine signaling. We conclude that CD177 signals in a β2 integrin-dependent manner to orchestrate a set of activation-mediated mechanisms that impair human neutrophil migration. © 2017 by The American Society of Hematology.
The small GTPase, Rap1, mediates CD31-induced integrin adhesion
Reedquist, K. A.; Ross, E.; Koop, E. A.; Wolthuis, R. M.; Zwartkruis, F. J.; van Kooyk, Y.; Salmon, M.; Buckley, C. D.; Bos, J. L.
2000-01-01
Integrin-mediated leukocyte adhesion is a critical aspect of leukocyte function that is tightly regulated by diverse stimuli, including chemokines, antigen receptors, and adhesion receptors. How cellular signals from CD31 and other adhesion amplifiers are integrated with those from classical
DEFF Research Database (Denmark)
Gustavsson, Anna; Armulik, Annika; Brakebusch, Cord
2002-01-01
Invasin of Yersinia pseudotuberculosis binds to beta1-integrins on host cells and triggers internalization of the bacterium. To elucidate the mechanism behind the beta1-integrin-mediated internalization of Yersinia, a beta1-integrin-deficient cell line, GD25, transfected with wild-type beta1A, beta......1B or different mutants of the beta1A subunit was used. Both beta1A and beta1B bound to invasin-expressing bacteria, but only beta1A was able to mediate internalization of the bacteria. The cytoplasmic region of beta1A, differing from beta1B, contains two NPXY motifs surrounding a double threonine...... noted that cells affected in bacterial internalization exhibited reduced spreading capability when seeded onto invasin, suggesting a correlation between the internalization of invasin-expressing bacteria and invasin-induced spreading. Likewise, integrins defective in forming peripheral focal complex...
Rossaint, Jan; Herter, Jan M.; van Aken, Hugo; Napirei, Markus; Döring, Yvonne; Weber, Christian; Soehnlein, Oliver; Zarbock, Alexander
2014-01-01
There is emerging evidence that neutrophil extracellular traps (NETs) play important roles in inflammatory processes. Here we report that neutrophils have to be simultaneously activated by integrin-mediated outside-in- and G-protein-coupled receptor (GPCR) signaling to induce NET formation in acute
Vesicle-associated membrane protein 2 mediates trafficking of α5β1 integrin to the plasma membrane
International Nuclear Information System (INIS)
Hasan, Nazarul; Hu, Chuan
2010-01-01
Integrins are major receptors for cell adhesion to the extracellular matrix (ECM). As transmembrane proteins, the levels of integrins at the plasma membrane or the cell surface are ultimately determined by the balance between two vesicle trafficking events: endocytosis of integrins at the plasma membrane and exocytosis of the vesicles that transport integrins. Here, we report that vesicle-associated membrane protein 2 (VAMP2), a SNARE protein that mediates vesicle fusion with the plasma membrane, is involved in the trafficking of α5β1 integrin. VAMP2 was present on vesicles containing endocytosed β1 integrin. Small interfering RNA (siRNA) silencing of VAMP2 markedly reduced cell surface α5β1 and inhibited cell adhesion and chemotactic migration to fibronectin, the ECM ligand of α5β1, without altering cell surface expression of α2β1 integrin or α3β1 integrin. By contrast, silencing of VAMP8, another SNARE protein, had no effect on cell surface expression of the integrins or cell adhesion to fibronectin. In addition, VAMP2-mediated trafficking is involved in cell adhesion to collagen but not to laminin. Consistent with disruption of integrin functions in cell proliferation and survival, VAMP2 silencing diminished proliferation and triggered apoptosis. Collectively, these data indicate that VAMP2 mediates the trafficking of α5β1 integrin to the plasma membrane and VAMP2-dependent integrin trafficking is critical in cell adhesion, migration and survival.
Sriramula, Srinivas; Lazartigues, Eric
2017-12-01
Hypertension is associated with increased activity of the kallikrein-kinin system. Kinin B1 receptor (B1R) activation leads to vasoconstriction and inflammation. Despite evidence supporting a role for the B1R in blood pressure regulation, the mechanisms by which B1R could alter autonomic function and participate in the pathogenesis of hypertension remain unidentified. We sought to explore whether B1R-mediated inflammation contributes to hypertension and investigate the molecular mechanisms involved. In this study, we tested the hypothesis that activation of B1R in the brain is involved in the pathogenesis of hypertension, using the deoxycorticosterone acetate-salt model of neurogenic hypertension in wild-type and B1R knockout mice. Deoxycorticosterone acetate-salt treatment in wild-type mice led to significant increases in B1R mRNA and protein levels and bradykinin levels, enhanced gene expression of carboxypeptidase N supporting an increase in the B1R ligand, associated with enhanced blood pressure, inflammation, sympathoexcitation, autonomic dysfunction, and impaired baroreflex sensitivity, whereas these changes were blunted or prevented in B1R knockout mice. B1R stimulation was further shown to involve activation of the ASK1-JNK-ERK1/2 and NF-κB pathways in the brain. To dismiss potential developmental alterations in knockout mice, we further used B1R blockade selectively in the brain of wild-type mice. Supporting the central origin of this mechanism, intracerebroventricular infusion of a specific B1R antagonist, attenuated the deoxycorticosterone acetate-salt-induced increase in blood pressure in wild-type mice. Our data provide the first evidence of a central role for B1R-mediated inflammatory pathways in the pathogenesis of deoxycorticosterone acetate-salt hypertension and offer novel insights into possible B1R-targeted therapies for the treatment of neurogenic hypertension. © 2017 American Heart Association, Inc.
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Dániel Szili
Full Text Available B cell development and activation are regulated by combined signals mediated by the B cell receptor (BCR, receptors for the B-cell activating factor of the tumor necrosis factor family (BAFF-R and the innate receptor, Toll-like receptor 9 (TLR9. However, the underlying mechanisms by which these signals cooperate in human B cells remain unclear. Our aim was to elucidate the key signaling molecules at the crossroads of BCR, BAFF-R and TLR9 mediated pathways and to follow the functional consequences of costimulation.Therefore we stimulated purified human B cells by combinations of anti-Ig, B-cell activating factor of the tumor necrosis factor family (BAFF and the TLR9 agonist, CpG oligodeoxynucleotide. Phosphorylation status of various signaling molecules, B cell proliferation, cytokine secretion, plasma blast generation and the frequency of IgG producing cells were investigated. We have found that BCR induced signals cooperate with BAFF-R- and TLR9-mediated signals at different levels of cell activation. BCR and BAFF- as well as TLR9 and BAFF-mediated signals cooperate at NFκB activation, while BCR and TLR9 synergistically costimulate mitogen activated protein kinases (MAPKs, ERK, JNK and p38. We show here for the first time that the MAP3K7 (TGF beta activated kinase, TAK1 is responsible for the synergistic costimulation of B cells by BCR and TLR9, resulting in an enhanced cell proliferation, plasma blast generation, cytokine and antibody production. Specific inhibitor of TAK1 as well as knocking down TAK1 by siRNA abrogates the synergistic signals. We conclude that TAK1 is a key regulator of receptor crosstalk between BCR and TLR9, thus plays a critical role in B cell development and activation.
Functions of Tenascin-C and Integrin alpha9beta1 in Mediating Prostate Cancer Bone Metastasis
2017-10-01
AWARD NUMBER: W81XWH-16-1-0523 TITLE: Functions of Tenascin- C and Integrin alpha9beta1 in Mediating Prostate Cancer Bone Metastasis...Prostat Prostate Cancer Bone Metastasis 5a. CONTRACT NUMBER Functions of Tenascin- C and Integrin alpha9beta1 in Mediating Prostate Cancer...SUPPLEMENTARY NOTES 14. ABSTRACT The purpose of this work is to dissect mechanisms responsible for interactions between integrin a9b1 and tenascin- C that are
Integrin β1 regulates leiomyoma cytoskeletal integrity and growth
Malik, Minnie; Segars, James; Catherino, William H.
2014-01-01
Uterine leiomyomas are characterized by an excessive extracellular matrix, increased mechanical stress, and increased active RhoA. Previously, we observed that mechanical signaling was attenuated in leiomyoma, but the mechanisms responsible remain unclear. Integrins, especially integrin β1, are transmembrane adhesion receptors that couple extracellular matrix stresses to the intracellular cytoskeleton to influence cell proliferation and differentiation. Here we characterized integrin and laminin to signaling in leiomyoma cells. We observed a 2.25 ± 0.32 fold increased expression of integrin β1 in leiomyoma cells, compared to myometrial cells. Antibody-mediated inhibition of integrin β1 led to significant growth inhibition in leiomyoma cells and a loss of cytoskeletal integrity. Specifically, polymerization of actin filaments and formation of focal adhesions were reduced by inhibition of integrin p1. Inhibition of integrin β1 in leiomyoma cells led to 0.81 ± 0.02 fold decrease in active RhoA, and resembled levels found in serum-starved cells. Likewise, inhibition of integrin β1 was accompanied by a decrease in phospho-ERK. Compared to myometrial cells, leiomyoma cells demonstrated increased expression of integrin α6 subunit to laminin receptor (1.91 ± 0.11 fold), and increased expression of laminin 5α (1.52±0.02), laminin 5β (3.06±0.92), and laminin 5γ (1.66 ± 0.06). Of note, leiomyoma cells grown on laminin matrix appear to realign themselves. Taken together, the findings reveal that the attenuated mechanical signaling in leiomyoma cells is accompanied by an increased expression and a dependence on integrin β1 signaling in leiomyoma cells, compared to myometrial cells. PMID:23023061
Beta1 integrin-mediated adhesion signalling is essential for epidermal progenitor cell expansion.
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Aleksandra Piwko-Czuchra
Full Text Available BACKGROUND: There is a major discrepancy between the in vitro and in vivo results regarding the role of beta1 integrins in the maintenance of epidermal stem/progenitor cells. Studies of mice with skin-specific ablation of beta1 integrins suggested that epidermis can form and be maintained in their absence, while in vitro data have shown a fundamental role for these adhesion receptors in stem/progenitor cell expansion and differentiation. METHODOLOGY/PRINCIPAL FINDINGS: To elucidate this discrepancy we generated hypomorphic mice expressing reduced beta1 integrin levels on keratinocytes that developed similar, but less severe defects than mice with beta1-deficient keratinocytes. Surprisingly we found that upon aging these abnormalities attenuated due to a rapid expansion of cells, which escaped or compensated for the down-regulation of beta1 integrin expression. A similar phenomenon was observed in aged mice with a complete, skin-specific ablation of the beta1 integrin gene, where cells that escaped Cre-mediated recombination repopulated the mutant skin in a very short time period. The expansion of beta1 integrin expressing keratinocytes was even further accelerated in situations of increased keratinocyte proliferation such as wound healing. CONCLUSIONS/SIGNIFICANCE: These data demonstrate that expression of beta1 integrins is critically important for the expansion of epidermal progenitor cells to maintain epidermal homeostasis.
Small molecule antagonists of integrin receptors.
Perdih, A; Dolenc, M Sollner
2010-01-01
The complex and widespread family of integrin receptors is involved in numerous physiological processes, such as tissue remodeling, angiogenesis, development of the immune response and homeostasis. In addition, their key role has been elucidated in important pathological disorders such as cancer, cardiovascular diseases, osteoporosis, autoimmune and inflammatory diseases and in the pathogenesis of infectious diseases, making them highly important targets for modern drug design campaigns. In this review we seek to present a concise overview of the small molecule antagonists of this diverse and highly complex receptor family. Integrin antagonists are classified according to the targeted integrin receptor and are discussed in four sections. First we present the fibrinogen alpha(IIb)beta3 and the vitronectin alpha (V)beta(3) receptor antagonists. The remaining selective integrin antagonists are examined in the third section. The final section is dedicated to molecules with dual or multiple integrin activity. In addition, the use of antibodies and peptidomimetic approaches to modulate the integrin receptors are discussed, as well providing the reader with an overall appreciation of the field.
Activated integrin VLA-4 localizes to the lamellipodia and mediates T cell migration on VCAM-11
Hyun, Young-Min; Chung, Hung-Li; McGrath, James L.; Waugh, Richard E.; Kim, Minsoo
2009-01-01
Lymphocyte migration from blood into lymphoid tissues or to sites of inflammation occurs through interactions between cell surface integrins and their ligands expressed on the vascular endothelium and the extracellular matrix. Very Late Antigen-4 (VLA-4, α4β1) is a key integrin in the effective trafficking of lymphocytes. Although it has been well established that integrins undergo functionally significant conformational changes to mediate cell adhesion, there is no mechanistic information that explains how these are dynamically and spatially regulated during lymphocyte polarization and migration. Using dynamic fluorescence resonance energy transfer (FRET) analysis of a novel VLA-4 FRET sensor under total internal reflection fluorescence (TIRF) microscopy, we show that VLA-4 activation localizes to the lamellipodium in living cells. During T cell migration on VCAM-1, VLA-4 activation concurs with spatial redistribution of chemokine receptor and active Rap1 at the leading edge. Selective inhibition of the activated VLA-4 at leading edge with a small molecule inhibitor is sufficient to block T cell migration. These data suggest that a subpopulation of activated VLA-4 is mainly localized to the leading edge of polarized human T cells, and is critical for T cell migration on VCAM-1. PMID:19542447
International Nuclear Information System (INIS)
Ruseva, Zlatna; Geiger, Pamina Xenia Charlotte; Hutzler, Peter; Kotzsch, Matthias; Luber, Birgit; Schmitt, Manfred; Gross, Eva; Reuning, Ute
2009-01-01
The tetraspanin KAI1 had been described as a metastasis suppressor in many different cancer types, a function for which associations of KAI1 with adhesion and signaling receptors of the integrin superfamily likely play a role. In ovarian cancer, integrin αvβ3 correlates with tumor progression and its elevation in vitro provoked enhanced cell adhesion accompanied by significant increases in cell motility and proliferation in the presence of its major ligand vitronectin. In the present study, we characterized integrin αvβ3-mediated tumor biological effects as a function of cellular KAI1 restoration and proved for the first time that KAI1, besides its already known physical crosstalk with β1-integrins, also colocalizes with integrin αvβ3. Functionally, elevated KAI1 levels drastically increased integrin αvβ3/vitronectin-dependent ovarian cancer cell adhesion. Since an intermediate level of cell adhesive strength is required for optimal cell migration, we next studied ovarian cancer cell motility as a function of KAI1 restoration. By time lapse video microscopy, we found impaired integrin αvβ3/vitronectin-mediated cell migration most probably due to strongly enhanced cellular immobilization onto the adhesion-supporting matrix. Moreover, KAI1 reexpression significantly diminished cell proliferation. These data strongly indicate that KAI1 may suppress ovarian cancer progression by inhibiting integrin αvβ3/vitronectin-provoked tumor cell motility and proliferation as important hallmarks of the oncogenic process.
Horiguchi, Kotaro; Fujiwara, Ken; Ilmiawati, Cimi; Kikuchi, Motoshi; Tsukada, Takehiro; Kouki, Tom; Yashiro, Takashi
2011-07-01
Folliculostellate (FS) cells in the anterior pituitary gland are believed to have multifunctional properties. Using transgenic rats that express green fluorescent protein (GFP) specifically in FS cells in the anterior pituitary gland (S100b-GFP rats), we recently revealed that FS cells in primary culture exhibited marked proliferation in the presence of laminin, an extracellular matrix (ECM) component of the basement membrane. In a process referred to as matricrine action, FS cells receive ECM as a signal through their receptors, which results in morphological and functional changes. In this study, we investigated matricrine signaling in FS cells and observed that the proliferation of FS cells is mediated by integrin β1, which is involved in various signaling pathways for cell migration and proliferation in response to ECM. Then, we analyzed downstream events of the integrin β1 signaling pathway in the proliferation of FS cells and identified caveolin 3 as a potential candidate molecule. Caveolin 3 is a membrane protein that binds cholesterol and a number of signaling molecules that interact with integrin β1. Using specific small interfering RNA of caveolin 3, the proliferation of FS cells was inhibited. Furthermore, caveolin 3 drove activation of the mitogen-activated protein kinase (MAPK) signaling cascades, which resulted in upregulation of cyclin D1 in FS cells. These findings suggest that matricrine signaling in the proliferation of FS cells was transduced by a caveolin 3-mediated integrin β1 signaling pathway and subsequent activation of the MAPK pathway. © 2011 Society for Endocrinology
Lu, Ling; Lin, ChangDong; Yan, ZhanJun; Wang, Shu; Zhang, YouHua; Wang, ShiHui; Wang, JunLei; Liu, Cui; Chen, JianFeng
2016-05-06
Integrin-mediated rolling and firm cell adhesion are two critical steps in leukocyte trafficking. Integrin α4β1 mediates a mixture of rolling and firm cell adhesion on vascular cell adhesion molecule-1 (VCAM-1) when in its resting state but only supports firm cell adhesion upon activation. The transition from rolling to firm cell adhesion is controlled by integrin activation. Kindlin-3 has been shown to bind to integrin β tails and trigger integrin activation via inside-out signaling. However, the role of kindlin-3 in regulating resting α4β1-mediated cell adhesion is not well characterized. Herein we demonstrate that kindlin-3 was required for the resting α4β1-mediated firm cell adhesion but not rolling adhesion. Knockdown of kindlin-3 significantly decreased the binding of kindlin-3 to β1 and down-regulated the binding affinity of the resting α4β1 to soluble VCAM-1. Notably, it converted the resting α4β1-mediated firm cell adhesion to rolling adhesion on VCAM-1 substrates, increased cell rolling velocity, and impaired the stability of cell adhesion. By contrast, firm cell adhesion mediated by Mn(2+)-activated α4β1 was barely affected by knockdown of kindlin-3. Structurally, lack of kindlin-3 led to a more bent conformation of the resting α4β1. Thus, kindlin-3 plays an important role in maintaining a proper conformation of the resting α4β1 to mediate both rolling and firm cell adhesion. Defective kindlin-3 binding to the resting α4β1 leads to a transition from firm to rolling cell adhesion on VCAM-1, implying its potential role in regulating the transition between integrin-mediated rolling and firm cell adhesion. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
Lu, Ling; Lin, ChangDong; Yan, ZhanJun; Wang, Shu; Zhang, YouHua; Wang, ShiHui; Wang, JunLei; Liu, Cui; Chen, JianFeng
2016-01-01
Integrin-mediated rolling and firm cell adhesion are two critical steps in leukocyte trafficking. Integrin α4β1 mediates a mixture of rolling and firm cell adhesion on vascular cell adhesion molecule-1 (VCAM-1) when in its resting state but only supports firm cell adhesion upon activation. The transition from rolling to firm cell adhesion is controlled by integrin activation. Kindlin-3 has been shown to bind to integrin β tails and trigger integrin activation via inside-out signaling. However, the role of kindlin-3 in regulating resting α4β1-mediated cell adhesion is not well characterized. Herein we demonstrate that kindlin-3 was required for the resting α4β1-mediated firm cell adhesion but not rolling adhesion. Knockdown of kindlin-3 significantly decreased the binding of kindlin-3 to β1 and down-regulated the binding affinity of the resting α4β1 to soluble VCAM-1. Notably, it converted the resting α4β1-mediated firm cell adhesion to rolling adhesion on VCAM-1 substrates, increased cell rolling velocity, and impaired the stability of cell adhesion. By contrast, firm cell adhesion mediated by Mn2+-activated α4β1 was barely affected by knockdown of kindlin-3. Structurally, lack of kindlin-3 led to a more bent conformation of the resting α4β1. Thus, kindlin-3 plays an important role in maintaining a proper conformation of the resting α4β1 to mediate both rolling and firm cell adhesion. Defective kindlin-3 binding to the resting α4β1 leads to a transition from firm to rolling cell adhesion on VCAM-1, implying its potential role in regulating the transition between integrin-mediated rolling and firm cell adhesion. PMID:26994136
Energy Technology Data Exchange (ETDEWEB)
Baek, Jong Min; Park, Sun-Hyang; Cheon, Yoon-Hee; Ahn, Sung-Jun [Department of Anatomy, School of Medicine, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Lee, Myeung Su [Division of Rheumatology, Department of Internal Medicine, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Imaging Science-based Lung and Bone Diseases Research Center, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Institute for Skeletal Disease, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Oh, Jaemin, E-mail: jmoh@wku.ac.kr [Department of Anatomy, School of Medicine, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Imaging Science-based Lung and Bone Diseases Research Center, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Institute for Skeletal Disease, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Kim, Ju-Young, E-mail: kimjy1014@gmail.com [Imaging Science-based Lung and Bone Diseases Research Center, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of)
2015-05-29
Esculetin exerts various biological effects on anti-oxidation, anti-tumors, and anti-inflammation. However, the involvement of esculetin in the bone metabolism process, particularly osteoclast differentiation has not yet been investigated. In the present study, we first confirmed the inhibitory effect of esculetin on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation. We then revealed the relationship between esculetin and the expression of osteoclast-specific molecules to elucidate its underlying mechanisms. Esculetin interfered with the expression of c-Fos and nuclear factor of activated T cell c1 (NFATc1) both at the mRNA and protein level with no involvement in osteoclast-associated early signaling pathways, suppressing the expression of various transcription factors exclusively expressed in osteoclasts such as tartrate-resistant acid phosphatase (Trap), osteoclast-associated receptor (Oscar), dendritic cell-specific transmembrane protein (Dcstamp), osteoclast stimulatory transmembrane protein (Ocstamp), cathepsin K, αvβ3 integrin, and calcitonin receptor (Ctr). Additionally, esculetin inhibited the formation of filamentous actin (F-actin) ring-positive osteoclasts during osteoclast differentiation. However, the development of F-actin structures and subsequent bone resorbing activity of mature osteoclasts, which are observed in osteoclast/osteoblast co-culture systems were not affected by esculetin. Taken together, our results indicate for the first time that esculetin inhibits RANKL-mediated osteoclastogenesis via direct suppression of c-Fos and NFATc1 expression and exerts an inhibitory effect on actin ring formation during osteoclastogenesis. - Highlights: • We first investigated the effects of esculetin on osteoclast differentiation and function. • Our data demonstrate for the first time that esculetin can suppress osteoclastogenesis in vitro. • Esculetin acts as an inhibitor of c-Fos and NFATc1 activation.
Phospho-Caveolin-1 Mediates Integrin-Regulated Membrane Domain Internalisation
del Pozo, Miguel A.; Alderson, Nazilla B.; Grande-García, Araceli; Balasubramanian, Nagaraj; Schwartz, Martin A.; Kiosses, William B.; Anderson, Richard G.W.
2005-01-01
Growth of normal cells is anchorage-dependent because signalling through multiple pathways including Erk, PI 3-kinase and Rac requires integrin-mediated cell adhesion 1. Components of these pathways localize to low density, cholesterol-rich domains in the plasma membrane named “lipid rafts” 2,3 or “cholesterol enriched membrane microdomains” (CEMM) 4. We previously reported that integrin-mediated adhesion regulates CEMM trafficking such that cell detachment from the extracellular matrix (ECM) triggers CEMM internalisation and clearance from the plasma membrane 5. We now report that this internalisation is mediated by dynamin-2 and caveolin-1. Internalisation requires phosphorylation of caveolin-1 on tyrosine 14. A shift in localisation of phospho-caveolin-1 from focal adhesions to caveolae induces CEMM internalisation upon cell detachment, which mediates inhibition of Erk, PI 3-kinase and Rac. These data define a novel molecular mechanism for growth and tumour suppression by caveolin-1. PMID:16113676
DEFF Research Database (Denmark)
Seidler, Julia; Durzok, Rita; Brakebusch, Cord
2005-01-01
25beta1B cells, which express mutant beta1B-integrins, were compared in terms of radiation survival and beta1-integrin signaling. MATERIALS AND METHODS: Cells grown on fibronectin, collagen-III, laminin, vitronectin, anti-beta1-integrin-IgG (beta1-IgG) or poly-l-lysine were irradiated with 0-6Gy...... and phosphorylation were analyzed by Western blot technique. RESULTS: Adhesion of GD25beta1A cells to extracellular matrix proteins or beta1-IgG resulted in growth factor-independent radiation survival. In contrast, serum starved GD25beta1B cells showed a significant (Pradiation survival on all...... phosphorylation. Phosphorylated p130Cas and paxillin subsequently prevented activation of cell death-regulating JNK. CONCLUSIONS: The data show that beta1-integrin-mediated signaling through the cytoplasmic integrin domains is critical for efficient pro-survival regulation after irradiation. Profound knowledge...
EMMPRIN regulates β1 integrin-mediated adhesion through Kindlin-3 in human melanoma cells.
Delyon, Julie; Khayati, Farah; Djaafri, Ibtissem; Podgorniak, Marie-Pierre; Sadoux, Aurélie; Setterblad, Niclas; Boutalbi, Zineb; Maouche, Kamel; Maskos, Uwe; Menashi, Suzanne; Lebbé, Céleste; Mourah, Samia
2015-06-01
EMMPRIN is known to promote tumor invasion through extracellular matrix (ECM) degradation. Here we report that EMMPRIN can regulate melanoma cell adhesion to the ECM through an interaction with β1 integrin involving kindlin-3. In this study, EMMPRIN knockdown in the human melanoma cell line M10 using siRNA decreased cell invasion and significantly increased cell adhesion and spreading. A morphological change from a round to a spread shape was observed associated with enhanced phalloidin-labelled actin staining. In situ proximity ligation assay and co-immunoprecipitation revealed that EMMPRIN silencing increased the interaction of β1 integrin with kindlin-3, a focal adhesion protein. This was associated with an increase in β1 integrin activation and a decrease in the phosphorylation of the downstream integrin kinase FAK. Moreover, the expression at both the transcript and protein level of kindlin-3 and of β1 integrin was inversely regulated by EMMPRIN. EMMPRIN did not regulate either talin expression or its interaction with β1 integrin. These results are consistent with our in vivo demonstration that EMMPRIN inhibition increased β1 integrin activation and its interaction with kindlin-3. To conclude, these findings reveal a new role of EMMPRIN in tumor cell migration through ß1 integrin/kindlin-3-mediated adhesion pathway. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Beauchemin, Hugues; Shooshtarizadeh, Peiman; Vadnais, Charles; Vassen, Lothar; Pastore, Yves D; Möröy, Tarik
2017-03-01
Mutations in GFI1B are associated with inherited bleeding disorders called GFI1B -related thrombocytopenias. We show here that mice with a megakaryocyte-specific Gfi1b deletion exhibit a macrothrombocytopenic phenotype along a megakaryocytic dysplasia reminiscent of GFI1B -related thrombocytopenia. GFI1B deficiency increases megakaryocyte proliferation and affects their ploidy, but also abrogates their responsiveness towards integrin signaling and their ability to spread and reorganize their cytoskeleton. Gfi1b -null megakaryocytes are also unable to form proplatelets, a process independent of integrin signaling. GFI1B-deficient megakaryocytes exhibit aberrant expression of several components of both the actin and microtubule cytoskeleton, with a dramatic reduction of α-tubulin. Inhibition of FAK or ROCK, both important for actin cytoskeleton organization and integrin signaling, only partially restored their response to integrin ligands, but the inhibition of PAK, a regulator of the actin cytoskeleton, completely rescued the responsiveness of Gfi1b -null megakaryocytes to ligands, but not their ability to form proplatelets. We conclude that Gfi1b controls major functions of megakaryocytes such as integrin-dependent cytoskeleton organization, spreading and migration through the regulation of PAK activity whereas the proplatelet formation defect in GFI1B-deficient megakaryocytes is due, at least partially, to an insufficient α-tubulin content. Copyright© Ferrata Storti Foundation.
Selective Modulation of Integrin-mediated Cell Migration by Distinct ADAM Family MembersV⃞
Huang, Jing; Bridges, Lance C.; White, Judith M.
2005-01-01
A disintegrin and a metalloprotease (ADAM) family members have been implicated in many biological processes. Although it is recognized that recombinant ADAM disintegrin domains can interact with integrins, little is known about ADAM-integrin interactions in cellular context. Here, we tested whether ADAMs can selectively regulate integrin-mediated cell migration. ADAMs were expressed in Chinese hamster ovary cells that express defined integrins (α4β1, α5β1, or both), and cell migration on full-length fibronectin or on its α4β1 or α5β1 binding fragments was studied. We found that ADAMs inhibit integrin-mediated cell migration in patterns dictated by the integrin binding profiles of their isolated disintegrin domains. ADAM12 inhibited cell migration mediated by the α4β1 but not the α5β1 integrin. ADAM17 had the reciprocal effect; it inhibited α5β1- but not α4β1-mediated cell migration. ADAM19 and ADAM33 inhibited migration mediated by both α4β1 and α5β1 integrins. A point mutation in the ADAM12 disintegrin loop partially reduced the inhibitory effect of ADAM12 on cell migration on the α4β1 binding fragment of fibronectin, whereas mutations that block metalloprotease activity had no effect. Our results indicate that distinct ADAMs can modulate cell migration mediated by specific integrins in a pattern dictated, at least in part, by their disintegrin domains. PMID:16079176
Directory of Open Access Journals (Sweden)
Alexandra Canonici
Full Text Available Intestinal epithelial cell damage is frequently seen in the mucosal lesions of infectious or inflammatory bowel diseases such as ulcerative colitis or Crohn's disease. Complete remission of these diseases requires both the disappearance of inflammation and the repair of damaged epithelium. Saccharomyces boulardii (Sb, Biocodex is a non-pathogenic yeast widely used as a preventive and therapeutic probiotic for the prevention and treatment of diarrhea and other gastrointestinal disorders. We recently showed that it enhances the repair of intestinal epithelium through activation of α2β1 integrin collagen receptors. In the present study, we demonstrated that α2β1 integrin is not the sole cell-extracellular matrix receptor involved during Sb-mediated intestinal restitution. Indeed, by using cell adhesion assays, we showed that Sb supernatant contains heat sensitive molecule(s, with a molecular weight higher than 9 kDa, which decreased αvβ5 integrin-mediated adhesion to vitronectin by competing with the integrin. Moreover, Sb-mediated changes in cell adhesion to vitronectin resulted in a reduction of the αvβ5signaling pathway. We used a monolayer wounding assay that mimics in vivo cell restitution to demonstrate that down-modulation of the αvβ5 integrin-vitronectin interaction is related to Sb-induced cell migration. We therefore postulated that Sb supernatant contains motogenic factors that enhance cell restitution through multiple pathways, including the dynamic fine regulation of αvβ5 integrin binding activity. This could be of major importance in diseases characterized by severe mucosal injury, such as inflammatory and infectious bowel diseases.
IGF-IR promotes prostate cancer growth by stabilizing α5β1 integrin protein levels.
Directory of Open Access Journals (Sweden)
Aejaz Sayeed
Full Text Available Dynamic crosstalk between growth factor receptors, cell adhesion molecules and extracellular matrix is essential for cancer cell migration and invasion. Integrins are transmembrane receptors that bind extracellular matrix proteins and enable cell adhesion and cytoskeletal organization. They also mediate signal transduction to regulate cell proliferation and survival. The type 1 insulin-like growth factor receptor (IGF-IR mediates tumor cell growth, adhesion and inhibition of apoptosis in several types of cancer. We have previously demonstrated that β1 integrins regulate anchorage-independent growth of prostate cancer (PrCa cells by regulating IGF-IR expression and androgen receptor-mediated transcriptional functions. Furthermore, we have recently reported that IGF-IR regulates the expression of β1 integrins in PrCa cells. We have dissected the mechanism through which IGF-IR regulates β1 integrin expression in PrCa. Here we report that IGF-IR is crucial for PrCa cell growth and that β1 integrins contribute to the regulation of proliferation by IGF-IR. We demonstrate that β1 integrin regulation by IGF-IR does not occur at the mRNA level. Exogenous expression of a CD4 - β1 integrin cytoplasmic domain chimera does not interfere with such regulation and fails to stabilize β1 integrin expression in the absence of IGF-IR. This appears to be due to the lack of interaction between the β1 cytoplasmic domain and IGF-IR. We demonstrate that IGF-IR stabilizes the β1 subunit by protecting it from proteasomal degradation. The α5 subunit, one of the binding partners of β1, is also downregulated along with β1 upon IGF-IR knockdown while no change is observed in the expression of the α2, α3, α4, α6 and α7 subunits. Our results reveal a crucial mechanistic role for the α5β1 integrin, downstream of IGF-IR, in regulating cancer growth.
Duan, Chaojun; Li, Minghua; Rui, Liangyou
2004-10-15
Leptin regulates energy homeostasis primarily by binding and activating its long form receptor (LRb). Deficiency of either leptin or LRb causes morbid obesity. Leptin stimulates LRb-associated JAK2, thus initiating multiple pathways including the Stat3 and phosphatidylinositol (PI) 3-kinase pathways that mediate leptin biological actions. Here we report that SH2-B, a JAK2-interacting protein, promotes activation of the PI 3-kinase pathway by recruiting insulin receptor substrate 1 (IRS1) and IRS2 in response to leptin. SH2-B directly bound, via its PH and SH2 domain, to both IRS1 and IRS2 both in vitro and in intact cells and mediated formation of a JAK2/SH2-B/IRS1 or IRS2 tertiary complex. Consequently, SH2-B dramatically enhanced leptin-stimulated tyrosine phosphorylation of IRS1 and IRS2 in HEK293 cells stably expressing LRb, thus promoting association of IRS1 and IRS2 with the p85 regulatory subunit of PI 3-kinase and phosphorylation and activation of Akt. SH2-B mutants with lower affinity for IRS1 and IRS2 exhibited reduced ability to promote association of JAK2 with IRS1, tyrosine phosphorylation of IRS1, and association of IRS1 with p85 in response to leptin. Moreover, deletion of the SH2-B gene impaired leptin-stimulated tyrosine phosphorylation of endogenous IRS1 in mouse embryonic fibroblasts (MEF), which was reversed by reintroduction of SH2-B. Similarly, SH2-B promoted growth hormone-stimulated tyrosine phosphorylation of IRS1 in both HEK293 and MEF cells. Our data suggest that SH2-B is a novel mediator of the PI 3-kinase pathway in response to leptin or other hormones and cytokines that activate JAK2.
Structural insight into the recognition of complement C3 activation products by integrin receptors
DEFF Research Database (Denmark)
Bajic, Goran
2015-01-01
fragment C3a called anaphylatoxin. Complement leads to opsonization as the proteolytic fragment C3b becomes covalently linked to the activator surface through a reactive thioester. Self-surfaces are protected by complement regulators, whereas complement activation vividly amplifies on pathogens...... and their clearance by dendritic cells is mediated by αMβ2. The central molecule in my project, αMβ2 integrin, recognizes many diverse ligands including iC3b, but the molecular basis for such recognition was lacking. During my PhD I have obtained a major breakthrough in the dissection of iC3b interaction with αMβ2. I...
Allain, Fabrice; Vanpouille, Christophe; Carpentier, Mathieu; Slomianny, Marie-Christine; Durieux, Sandrine; Spik, Geneviève
2002-03-05
Cyclophilins A and B (CyPA and CyPB) are cyclosporin A-binding proteins that are involved in inflammatory events. We have reported that CyPB interacts with two types of cell-surface-binding sites. The first site corresponds to a functional receptor and requires interaction with the central core of CyPB. This region is highly conserved in cyclophilins, suggesting that CyPA and CyPB might share biological activities mediated by interaction with this receptor. The second site is identified with glycosaminoglycans (GAGs), the binding region located in the N terminus of CyPB. The difference in the N-terminal extensions of CyPA and CyPB suggests that a unique interaction with GAGs might account for selective activity of CyPB. To explore this hypothesis, we analyzed the lymphocyte responses triggered by CyPA, CyPB, and CyPB(KKK-), a mutant unable to interact with GAGs. The three ligands seemed capable enough to elicit calcium signal and chemotaxis by binding to the same signaling receptor. In contrast, only CyPB enhanced firm adhesion of T cells to the extracellular matrix. This activity depended on the interactions with GAGs and signaling receptor. CyPB-mediated adhesion required CD147 presumably because it was a costimulatory molecule and was related to an activation of alpha4beta1 and alpha4beta7 integrins. Finally, we showed that CyPB was capable mainly to enhance T cell adhesion of the CD4+CD45RO+ subset. The present data indicate that CyPB rather than CyPA is a proinflammatory factor for T lymphocytes and highlight the crucial role of CyPB-GAG interaction in the chemokine-like activity of this protein.
Grove, Lisa M; Southern, Brian D; Jin, Tong H; White, Kimberly E; Paruchuri, Sailaja; Harel, Efrat; Wei, Ying; Rahaman, Shaik O; Gladson, Candece L; Ding, Qiang; Craik, Charles S; Chapman, Harold A; Olman, Mitchell A
2014-05-02
The urokinase-type plasminogen activator receptor (uPAR) is a glycosylphosphatidylinositol-linked membrane protein with no cytosolic domain that localizes to lipid raft microdomains. Our laboratory and others have documented that lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) exhibit a hypermotile phenotype. This study was undertaken to elucidate the molecular mechanism whereby uPAR ligation with its cognate ligand, urokinase, induces a motile phenotype in human lung fibroblasts. We found that uPAR ligation with the urokinase receptor binding domain (amino-terminal fragment) leads to enhanced migration of fibroblasts on fibronectin in a protease-independent, lipid raft-dependent manner. Ligation of uPAR with the amino-terminal fragment recruited α5β1 integrin and the acylated form of the Src family kinase, Fyn, to lipid rafts. The biological consequences of this translocation were an increase in fibroblast motility and a switch of the integrin-initiated signal pathway for migration away from the lipid raft-independent focal adhesion kinase pathway and toward a lipid raft-dependent caveolin-Fyn-Shc pathway. Furthermore, an integrin homologous peptide as well as an antibody that competes with β1 for uPAR binding have the ability to block this effect. In addition, its relative insensitivity to cholesterol depletion suggests that the interactions of α5β1 integrin and uPAR drive the translocation of α5β1 integrin-acylated Fyn signaling complexes into lipid rafts upon uPAR ligation through protein-protein interactions. This signal switch is a novel pathway leading to the hypermotile phenotype of IPF patient-derived fibroblasts, seen with uPAR ligation. This uPAR dependent, fibrotic matrix-selective, and profibrotic fibroblast phenotype may be amenable to targeted therapeutics designed to ameliorate IPF.
International Nuclear Information System (INIS)
Kikkawa, Yamato; Yu, Hao; Genersch, Elke; Sanzen, Noriko; Sekiguchi, Kiyotoshi; Faessler, Reinhard; Campbell, Kevin P.; Talts, Jan F.; Ekblom, Peter
2004-01-01
The presence of many laminin receptors of the β1 integrin family on most cells makes it difficult to define the biological functions of other major laminin receptors such as integrin α6β4 and dystroglycan. We therefore tested the binding of a β1 integrin-null cell line GD25 to four different laminin variants. The cells were shown to produce dystroglycan, which based on affinity chromatography bound to laminin-1, -2/4, and -10/11, but not to laminin-5. The cells also expressed the integrin α6Aβ4A variant. GD25 β1 integrin-null cells are known to bind poorly to laminin-1, but we demonstrate here that these cells bind avidly to laminin-2/4, -5, and -10/11. The initial binding at 20 min to each of these laminins could be inhibited by an integrin α6 antibody, but not by a dystroglycan antibody. Hence, integrin α6Aβ4A of GD25 cells was identified as a major receptor for initial GD25 cell adhesion to three out of four tested laminin isoforms. Remarkably, cell adhesion to laminin-5 failed to promote cell spreading, proliferation, and extracellular signal-regulated kinase (ERK) activation, whereas all these responses occurred in response to adhesion to laminin-2/4 or -10/11. The data establish GD25 cells as useful tools to define the role integrin α6Aβ4A and suggest that laminin isoforms have distinctly different capacities to promote cell adhesion and signaling via integrin α6Aβ4A
The membrane-cytoplasm interface of integrin alpha subunits is critical for receptor latency.
Briesewitz, R; Kern, A; Smilenov, L B; David, F S; Marcantonio, E E
1996-01-01
Localization of integrin receptors to focal contact sites occurs upon ligand binding. This activity is latent, since unoccupied integrin receptors do not localize to focal contacts. Deletion analysis has revealed that the alpha cytoplasmic domains is required for the maintenance of integrin receptor latency. Our current hypothesis for the mechanism of integrin post-ligand binding events is that there is a change in relationship of alpha and beta cytoplasmic domains, which overcomes receptor l...
Deuschle, Eva; Keller, Birgit; Siegfried, Alexandra; Manncke, Birgit; Spaeth, Tanja; Köberle, Martin; Drechsler-Hake, Doreen; Reber, Julia; Böttcher, Ralph T; Autenrieth, Stella E; Autenrieth, Ingo B; Bohn, Erwin; Schütz, Monika
2016-02-01
Injection of Yersinia outer proteins (Yops) into host cells by a type III secretion system is an important immune evasion mechanism of Yersinia enterocolitica (Ye). In this process Ye invasin (Inv) binds directly while Yersinia adhesin A (YadA) binds indirectly via extracellular matrix (ECM) proteins to β1 integrins on host cells. Although leukocytes turned out to be an important target of Yop injection by Ye, it was unclear which Ye adhesins and which leukocyte receptors are required for Yop injection. To explain this, we investigated the role of YadA, Inv and β1 integrins for Yop injection into leukocytes and their impact on the course of systemic Ye infection in mice. Ex vivo infection experiments revealed that adhesion of Ye via Inv or YadA is sufficient to promote Yop injection into leukocytes as revealed by a β-lactamase reporter assay. Serum factors inhibit YadA- but not Inv-mediated Yop injection into B and T cells, shifting YadA-mediated Yop injection in the direction of neutrophils and other myeloid cells. Systemic Ye mouse infection experiments demonstrated that YadA is essential for Ye virulence and Yop injection into leukocytes, while Inv is dispensable for virulence and plays only a transient and minor role for Yop injection in the early phase of infection. Ye infection of mice with β1 integrin-depleted leukocytes demonstrated that β1 integrins are dispensable for YadA-mediated Yop injection into leukocytes, but contribute to Inv-mediated Yop injection. Despite reduced Yop injection into leukocytes, β1 integrin-deficient mice exhibited an increased susceptibility for Ye infection, suggesting an important role of β1 integrins in immune defense against Ye. This study demonstrates that Yop injection into leukocytes by Ye is largely mediated by YadA exploiting, as yet unknown, leukocyte receptors. Copyright © 2015. Published by Elsevier GmbH.
LENUS (Irish Health Repository)
Wang, Jiang Huai
2012-02-03
Beta(1) integrins play a crucial role in supporting tumor cell attachment to and invasion into the extracellular matrix. Endotoxin\\/LPS introduced by surgery has been shown to enhance tumor metastasis in a murine model. Here we show the direct effect of LPS on tumor cell adhesion and invasion in extracellular matrix proteins through a beta(1) integrin-dependent pathway. The human colorectal tumor cell lines SW480 and SW620 constitutively expressed high levels of the beta(1) subunit, whereas various low levels of alpha(1), alpha(2), alpha(4), and alpha(6) expression were detected. SW480 and SW620 did not express membrane-bound CD14; however, LPS in the presence of soluble CD14 (sCD14) significantly up-regulated beta(1) integrin expression; enhanced tumor cell attachment to fibronectin, collagen I, and laminin; and strongly promoted tumor cell invasion through the Matrigel. Anti-beta(1) blocking mAbs (4B4 and 6S6) abrogated LPS- plus sCD14-induced tumor cell adhesion and invasion. Furthermore, LPS, when combined with sCD14, resulted in NF-kappaB activation in both SW480 and SW620 cells. Inhibition of the NF-kappaB pathway significantly attenuated LPS-induced up-regulation of beta(1) integrin expression and prevented tumor cell adhesion and invasion. These results provide direct evidence that although SW480 and SW620 cells do not express membrane-bound CD14, LPS in the presence of sCD14 can activate NF-kappaB, up-regulate beta(1) integrin expression, and subsequently promote tumor cell adhesion and invasion. Moreover, LPS-induced tumor cell attachment to and invasion through extracellular matrix proteins is beta(1) subunit-dependent.
The Integrin Receptor in Biologically Relevant Bilayers
DEFF Research Database (Denmark)
Kalli, Antreas C.; Róg, Tomasz; Vattulainen, Ilpo
2017-01-01
/talin complex was inserted in biologically relevant bilayers that resemble the cell plasma membrane containing zwitterionic and charged phospholipids, cholesterol and sphingolipids to study the dynamics of the integrin receptor and its effect on bilayer structure and dynamics. The results of this study...... demonstrate the dynamic nature of the integrin receptor and suggest that the presence of the integrin receptor alters the lipid organization between the two leaflets of the bilayer. In particular, our results suggest elevated density of cholesterol and of phosphatidylserine lipids around the integrin....../talin complex and a slowing down of lipids in an annulus of ~30 Å around the protein due to interactions between the lipids and the integrin/talin F2–F3 complex. This may in part regulate the interactions of integrins with other related proteins or integrin clustering thus facilitating signal transduction...
Kračun, Damir; Rieß, Florian; Kanchev, Ivan; Gawaz, Meinrad
2014-01-01
Abstract Aims: Integrins are multifunctional heterodimeric adhesion receptors that mediate the attachment between a cell and the extracellular matrix or other surrounding cells. In endothelial cells, integrins can modulate cell migration and motility. In particular, β3-integrin is expressed in angiogenic vessels. Signal transduction by β3-integrins requires the recruitment of intracellular signaling molecules. β3-endonexin is a highly spliced molecule that has been identified as a β3-integrin binding protein. β3-endonexin isoforms are expressed in endothelial cells and have been suggested to act as shuttle proteins between the membrane and the nucleus. However, their functional role in angiogenesis is unclear. In this study, we investigated whether β3-endonexin isoforms are involved in endothelial angiogenic processes under hypoxia. Results: The overexpression of β3-endonexin isoforms decreased endothelial proliferation and tube formation under hypoxia, while the depletion of β3-endonexin by RNAi promoted angiogenic responses in vitro and in vivo. In hypoxia, β3-endonexin accumulated in the nucleus, and prevention of this response by depletion of β3-endonexin increased hypoxic activation and induction of the hypoxia-inducible factor (HIF)-1 and its target genes VEGF and PAI-1. β3-endonexin diminished nuclear factor kappa B (NFκB) activation and decreased NFκB binding to the HIF-1α promoter under hypoxia, subsequently diminishing NFκB-dependent transcription of HIF-1α under hypoxia. Innovation: Our results indicate for the first time that the overexpression of β3-endonexin can decrease hypoxic induction and activation of HIF-1α and can prevent hypoxic endothelial proliferation and angiogenic responses. Conclusion: β3-endonexin can act as a novel anti-angiogenic factor specifically in the response to hypoxia due to its negative impact on the activation of HIF-1. Antioxid. Redox Signal. 20, 1964–1976. PMID:24386901
β1-integrin controls cell fate specification in early lens development
Pathania, Mallika; Wang, Yan; Simirskii, Vladimir N.; Duncan, Melinda K.
2016-01-01
Integrins are heterodimeric cell surface molecules that mediate cell-extracellular matrix (ECM) adhesion, ECM assembly, and regulation of both ECM and growth factor induced signaling. However, the developmental context of these diverse functions is not clear. Loss of β1-integrin from the lens vesicle (mouse E10.5) results in abnormal exit of anterior lens epithelial cells (LECs) from the cell cycle and their aberrant elongation toward the presumptive cornea by E12.5. These cells lose expression of LEC markers and initiate expression of the Maf (also known as c-Maf) and Prox1 transcription factors as well as other lens fiber cell markers, β1-integrin null LECs also upregulate the ERK, AKT and Smad1/5/8 phosphorylation indicative of BMP and FGF signaling. By E14.5, β1-integrin null lenses have undergone a complete conversion of all lens epithelial cells into fiber cells. These data suggest that shortly after lens vesicle closure, β1-integrin blocks inappropriate differentiation of the lens epithelium into fibers, potentially by inhibiting BMP and/or FGF receptor activation. Thus, β1-integrin has an important role in fine-tuning the response of the early lens to the gradient of growth factors that regulate lens fiber cell differentiation. PMID:27596755
Retinoids induce integrin-independent lymphocyte adhesion through RAR-α nuclear receptor activity
International Nuclear Information System (INIS)
Whelan, Jarrett T.; Wang, Lei; Chen, Jianming; Metts, Meagan E.; Nasser, Taj A.; McGoldrick, Liam J.; Bridges, Lance C.
2014-01-01
Highlights: • Transcription and translation are required for retinoid-induced lymphocyte adhesion. • RAR activation is sufficient to induced lymphocyte cell adhesion. • Vitamin D derivatives inhibit RAR-prompted lymphocyte adhesion. • Adhesion occurs through a novel binding site within ADAM disintegrin domains. • RARα is a key nuclear receptor for retinoid-dependent lymphocyte cell adhesion. - Abstract: Oxidative metabolites of vitamin A, in particular all-trans-retinoic acid (atRA), have emerged as key factors in immunity by specifying the localization of immune cells to the gut. Although it is appreciated that isomers of retinoic acid activate the retinoic acid receptor (RAR) and retinoid X receptor (RXR) family of nuclear receptors to elicit cellular changes, the molecular details of retinoic acid action remain poorly defined in immune processes. Here we employ a battery of agonists and antagonists to delineate the specific nuclear receptors utilized by retinoids to evoke lymphocyte cell adhesion to ADAM (adisintegrin and metalloprotease) protein family members. We report that RAR agonism is sufficient to promote immune cell adhesion in both immortal and primary immune cells. Interestingly, adhesion occurs independent of integrin function, and mutant studies demonstrate that atRA-induced adhesion to ADAM members required a distinct binding interface(s) as compared to integrin recognition. Anti-inflammatory corticosteroids as well as 1,25-(OH) 2 D 3 , a vitamin D metabolite that prompts immune cell trafficking to the skin, potently inhibited the observed adhesion. Finally, our data establish that induced adhesion was specifically attributable to the RAR-α receptor isotype. The current study provides novel molecular resolution as to which nuclear receptors transduce retinoid exposure into immune cell adhesion
Retinoids induce integrin-independent lymphocyte adhesion through RAR-α nuclear receptor activity
Energy Technology Data Exchange (ETDEWEB)
Whelan, Jarrett T.; Wang, Lei; Chen, Jianming; Metts, Meagan E.; Nasser, Taj A.; McGoldrick, Liam J. [Department of Biochemistry and Molecular Biology, The Brody School of Medicine at East Carolina University, Greenville, NC 27834 (United States); Bridges, Lance C., E-mail: bridgesl@ecu.edu [Department of Biochemistry and Molecular Biology, The Brody School of Medicine at East Carolina University, Greenville, NC 27834 (United States); East Carolina Diabetes and Obesity Institute, The Brody School of Medicine at East Carolina University, Greenville, NC 27834 (United States)
2014-11-28
Highlights: • Transcription and translation are required for retinoid-induced lymphocyte adhesion. • RAR activation is sufficient to induced lymphocyte cell adhesion. • Vitamin D derivatives inhibit RAR-prompted lymphocyte adhesion. • Adhesion occurs through a novel binding site within ADAM disintegrin domains. • RARα is a key nuclear receptor for retinoid-dependent lymphocyte cell adhesion. - Abstract: Oxidative metabolites of vitamin A, in particular all-trans-retinoic acid (atRA), have emerged as key factors in immunity by specifying the localization of immune cells to the gut. Although it is appreciated that isomers of retinoic acid activate the retinoic acid receptor (RAR) and retinoid X receptor (RXR) family of nuclear receptors to elicit cellular changes, the molecular details of retinoic acid action remain poorly defined in immune processes. Here we employ a battery of agonists and antagonists to delineate the specific nuclear receptors utilized by retinoids to evoke lymphocyte cell adhesion to ADAM (adisintegrin and metalloprotease) protein family members. We report that RAR agonism is sufficient to promote immune cell adhesion in both immortal and primary immune cells. Interestingly, adhesion occurs independent of integrin function, and mutant studies demonstrate that atRA-induced adhesion to ADAM members required a distinct binding interface(s) as compared to integrin recognition. Anti-inflammatory corticosteroids as well as 1,25-(OH){sub 2}D{sub 3}, a vitamin D metabolite that prompts immune cell trafficking to the skin, potently inhibited the observed adhesion. Finally, our data establish that induced adhesion was specifically attributable to the RAR-α receptor isotype. The current study provides novel molecular resolution as to which nuclear receptors transduce retinoid exposure into immune cell adhesion.
Tringali, Cristina; Lupo, Barbara; Silvestri, Ilaria; Papini, Nadia; Anastasia, Luigi; Tettamanti, Guido; Venerando, Bruno
2012-01-01
The human plasma membrane sialidase NEU3 is a key enzyme in the catabolism of membrane gangliosides, is crucial in the regulation of cell surface processes, and has been demonstrated to be significantly up-regulated in renal cell carcinomas (RCCs). In this report, we show that NEU3 regulates β1 integrin trafficking in RCC cells by controlling β1 integrin recycling to the plasma membrane and controlling activation of the epidermal growth factor receptor (EGFR) and focal adhesion kinase (FAK)/protein kinase B (AKT) signaling. NEU3 silencing in RCC cells increased the membrane ganglioside content, in particular the GD1a content, and changed the expression of key regulators of the integrin recycling pathway. In addition, NEU3 silencing up-regulated the Ras-related protein RAB25, which directs internalized integrins to lysosomes, and down-regulated the chloride intracellular channel protein 3 (CLIC3), which induces the recycling of internalized integrins to the plasma membrane. In this manner, NEU3 silencing enhanced the caveolar endocytosis of β1 integrin, blocked its recycling and reduced its levels at the plasma membrane, and, consequently, inhibited EGFR and FAK/AKT. These events had the following effects on the behavior of RCC cells: they (a) decreased drug resistance mediated by the block of autophagy and the induction of apoptosis; (b) decreased metastatic potential mediated by down-regulation of the metalloproteinases MMP1 and MMP7; and (c) decreased adhesion to collagen and fibronectin. Therefore, our data identify NEU3 as a key regulator of the β1 integrin-recycling pathway and FAK/AKT signaling and demonstrate its crucial role in RCC malignancy. PMID:23139422
Polycystin-1 promotes PKCα-mediated NF-κB activation in kidney cells
International Nuclear Information System (INIS)
Banzi, Manuela; Aguiari, Gianluca; Trimi, Viky; Mangolini, Alessandra; Pinton, Paolo; Witzgall, Ralph; Rizzuto, Rosario; Senno, Laura del
2006-01-01
Polycystin-1 (PC1), the PKD1 gene product, is a membrane receptor which regulates many cell functions, including cell proliferation and apoptosis, both typically increased in cyst lining cells in autosomal dominant polycystic kidney disease. Here we show that PC1 upregulates the NF-κB signalling pathway in kidney cells to prevent cell death. Human embryonic kidney cell lines (HEK293 CTT ), stably expressing a PC1 cytoplasmic terminal tail (CTT), presented increased NF-κB nuclear levels and NF-κB-mediated luciferase promoter activity. This, consistently, was reduced in HEK293 cells in which the endogenous PC1 was depleted by RNA interference. CTT-dependent NF-κB promoter activation was mediated by PKCα because it was blocked by its specific inhibitor Ro-320432. Furthermore, it was observed that apoptosis, which was increased in PC1-depleted cells, was reduced in HEK293 CTT cells and in porcine kidney LtTA cells expressing a doxycycline-regulated CTT. Staurosporine, a PKC inhibitor, and parthenolide, a NF-κB inhibitor, significantly reduced the CTT-dependent antiapoptotic effect. These data reveal, therefore, a novel pathway by which polycystin-1 activates a PKCα-mediated NF-κB signalling and cell survival
Ho, Ernest; Dagnino, Lina
2012-01-01
Epidermal growth factor (EGF) is a potent chemotactic and mitogenic factor for epidermal keratinocytes, and these properties are central for normal epidermal regeneration after injury. The involvement of mitogen-activated protein kinases as mediators of the proliferative effects of EGF is well established. However, the molecular mechanisms that mediate motogenic responses to this growth factor are not clearly understood. An obligatory step for forward cell migration is the development of front–rear polarity and formation of lamellipodia at the leading edge. We show that stimulation of epidermal keratinocytes with EGF, but not with other growth factors, induces development of front–rear polarity and directional migration through a pathway that requires integrin-linked kinase (ILK), Engulfment and Cell Motility-2 (ELMO2), integrin β1, and Rac1. Furthermore, EGF induction of front–rear polarity and chemotaxis require the tyrosine kinase activity of the EGF receptor and are mediated by complexes containing active RhoG, ELMO2, and ILK. Our findings reveal a novel link between EGF receptor stimulation, ILK-containing complexes, and activation of small Rho GTPases necessary for acquisition of front–rear polarity and forward movement. PMID:22160594
Ho, Ernest; Dagnino, Lina
2012-02-01
Epidermal growth factor (EGF) is a potent chemotactic and mitogenic factor for epidermal keratinocytes, and these properties are central for normal epidermal regeneration after injury. The involvement of mitogen-activated protein kinases as mediators of the proliferative effects of EGF is well established. However, the molecular mechanisms that mediate motogenic responses to this growth factor are not clearly understood. An obligatory step for forward cell migration is the development of front-rear polarity and formation of lamellipodia at the leading edge. We show that stimulation of epidermal keratinocytes with EGF, but not with other growth factors, induces development of front-rear polarity and directional migration through a pathway that requires integrin-linked kinase (ILK), Engulfment and Cell Motility-2 (ELMO2), integrin β1, and Rac1. Furthermore, EGF induction of front-rear polarity and chemotaxis require the tyrosine kinase activity of the EGF receptor and are mediated by complexes containing active RhoG, ELMO2, and ILK. Our findings reveal a novel link between EGF receptor stimulation, ILK-containing complexes, and activation of small Rho GTPases necessary for acquisition of front-rear polarity and forward movement.
Lee, JeHoon; Banu, Sakhila K; Burghardt, Robert C; Starzinski-Powitz, Anna; Arosh, Joe A
2013-03-01
Endometriosis is a chronic gynecological disease of reproductive age women characterized by the presence of functional endometrial tissues outside the uterine cavity. Interactions between the endometriotic cells and the peritoneal extracellular matrix proteins (ECM) are crucial mechanisms that allow adhesion of the endometriotic cells into peritoneal mesothelia. Prostaglandin E2 (PGE2) plays an important role in the pathogenesis of endometriosis. In previous studies, we have reported that selective inhibition of PGE2 receptors PTGER2 and PTGER4 decreases survival and invasion of human endometriotic epithelial and stromal cells through multiple mechanisms. Results of the present study indicates that selective inhibition of PTGER2- and PTGER4-mediated PGE2 signaling 1) decreases the expression and/or activity of specific integrin receptor subunits Itgb1 (beta1) and Itgb3 (beta3) but not Itgb5 (beta5), Itga1 (alpha1), Itga2 (alpha2), Itga5 (alpha5), and Itgav (alphav); 2) decreases integrin-signaling components focal adhesion kinase or protein kinase 2 (PTK2) and talin proteins; 3) inhibits interactions between Itgb1/Itgb3 subunits, PTK2, and talin and PTGER2/PTGER4 proteins through beta-arrestin-1 and Src kinase protein complex in human endometriotic epithelial cells 12Z and stromal cells 22B; and 4) decreases adhesion of 12Z and 22B cells to ECM collagen I, collagen IV, fibronectin, and vitronectin in a substrate-specific manner. These novel findings provide an important molecular framework for further evaluation of selective inhibition of PTGER2 and PTGER4 as potential nonsteroidal therapy to expand the spectrum of currently available treatment options for endometriosis in child-bearing age women.
Beta1 integrin-mediated adhesion signalling is essential for epidermal progenitor cell expansion
DEFF Research Database (Denmark)
Piwko-Czuchra, Aleksandra; Koegel, Heidi; Meyer, Hannelore
2009-01-01
BACKGROUND: There is a major discrepancy between the in vitro and in vivo results regarding the role of beta1 integrins in the maintenance of epidermal stem/progenitor cells. Studies of mice with skin-specific ablation of beta1 integrins suggested that epidermis can form and be maintained in thei...... of increased keratinocyte proliferation such as wound healing. CONCLUSIONS/SIGNIFICANCE: These data demonstrate that expression of beta1 integrins is critically important for the expansion of epidermal progenitor cells to maintain epidermal homeostasis....... that developed similar, but less severe defects than mice with beta1-deficient keratinocytes. Surprisingly we found that upon aging these abnormalities attenuated due to a rapid expansion of cells, which escaped or compensated for the down-regulation of beta1 integrin expression. A similar phenomenon...... was observed in aged mice with a complete, skin-specific ablation of the beta1 integrin gene, where cells that escaped Cre-mediated recombination repopulated the mutant skin in a very short time period. The expansion of beta1 integrin expressing keratinocytes was even further accelerated in situations...
Integrins and extracellular matrix in mechanotransduction
Directory of Open Access Journals (Sweden)
Ramage L
2011-12-01
Full Text Available Lindsay RamageQueen’s Medical Research Institute, University of Edinburgh, Edinburgh, UKAbstract: Integrins are a family of cell surface receptors which mediate cell–matrix and cell–cell adhesions. Among other functions they provide an important mechanical link between the cells external and intracellular environments while the adhesions that they form also have critical roles in cellular signal-transduction. Cell–matrix contacts occur at zones in the cell surface where adhesion receptors cluster and when activated the receptors bind to ligands in the extracellular matrix. The extracellular matrix surrounds the cells of tissues and forms the structural support of tissue which is particularly important in connective tissues. Cells attach to the extracellular matrix through specific cell-surface receptors and molecules including integrins and transmembrane proteoglycans. Integrins work alongside other proteins such as cadherins, immunoglobulin superfamily cell adhesion molecules, selectins, and syndecans to mediate cell–cell and cell–matrix interactions and communication. Activation of adhesion receptors triggers the formation of matrix contacts in which bound matrix components, adhesion receptors, and associated intracellular cytoskeletal and signaling molecules form large functional, localized multiprotein complexes. Cell–matrix contacts are important in a variety of different cell and tissue properties including embryonic development, inflammatory responses, wound healing, and adult tissue homeostasis. This review summarizes the roles and functions of integrins and extracellular matrix proteins in mechanotransduction.Keywords: ligand binding, α subunit, ß subunit, focal adhesion, cell differentiation, mechanical loading, cell–matrix interaction
Energy Technology Data Exchange (ETDEWEB)
Lochter, Andre; Navre, Marc; Werb, Zena; Bissell, Mina J
1998-06-29
Tumor cell invasion relies on cell migration and extracellular matrix proteolysis. We investigated the contribution of different integrins to the invasive activity of mouse mammary carcinoma cells. Antibodies against integrin subunits {alpha}6 and {beta}1, but not against {alpha}1 and {alpha}2, inhibited cell locomotion on a reconstituted basement membrane in two-dimensional cell migration assays, whereas antibodies against {beta}1, but not against a6 or {alpha}2, interfered with cell adhesion to basement membrane constituents. Blocking antibodies against {alpha}1 integrins impaired only cell adhesion to type IV collagen. Antibodies against {alpha}1, {alpha}2, {alpha}6, and {beta}1, but not {alpha}5, integrin subunits reduced invasion of a reconstituted basement membrane. Integrins {alpha}1 and {alpha}2, which contributed only marginally to motility and adhesion, regulated proteinase production. Antibodies against {alpha}1 and {alpha}2, but not {alpha}6 and {beta}1, integrin subunits inhibited both transcription and protein expression of the matrix metalloproteinase stromelysin-1. Inhibition of tumor cell invasion by antibodies against {alpha}1 and {alpha}2 was reversed by addition of recombinant stromelysin-1. In contrast, stromelysin-1 could not rescue invasion inhibited by anti-{alpha}6 antibodies. Our data indicate that {alpha}1 and {alpha}2 integrins confer invasive behavior by regulating stromelysin-1 expression, whereas {alpha}6 integrins regulate cell motility. These results provide new insights into the specific functions of integrins during tumor cell invasion.
Integrin α5β1, the Fibronectin Receptor, as a Pertinent Therapeutic Target in Solid Tumors
Energy Technology Data Exchange (ETDEWEB)
Schaffner, Florence; Ray, Anne Marie; Dontenwill, Monique, E-mail: monique.dontenwill@unistra.fr [UMR 7213 CNRS, Laboratoire de Biophotonique et Pharmacologie, Tumoral signaling and therapeutic targets, Université de Strasbourg, Faculté de Pharmacie, 67401 Illkirch (France)
2013-01-15
Integrins are transmembrane heterodimeric proteins sensing the cell microenvironment and modulating numerous signalling pathways. Changes in integrin expression between normal and tumoral cells support involvement of specific integrins in tumor progression and aggressiveness. This review highlights the current knowledge about α5β1 integrin, also called the fibronectin receptor, in solid tumors. We summarize data showing that α5β1 integrin is a pertinent therapeutic target expressed by tumoral neovessels and tumoral cells. Although mainly evaluated in preclinical models, α5β1 integrin merits interest in particular in colon, breast, ovarian, lung and brain tumors where its overexpression is associated with a poor prognosis for patients. Specific α5β1 integrin antagonists will be listed that may represent new potential therapeutic agents to fight defined subpopulations of particularly aggressive tumors.
Beyond the Matrix: The Many Non-ECM Ligands for Integrins
Directory of Open Access Journals (Sweden)
Bryce LaFoya
2018-02-01
Full Text Available The traditional view of integrins portrays these highly conserved cell surface receptors as mediators of cellular attachment to the extracellular matrix (ECM, and to a lesser degree, as coordinators of leukocyte adhesion to the endothelium. These canonical activities are indispensable; however, there is also a wide variety of integrin functions mediated by non-ECM ligands that transcend the traditional roles of integrins. Some of these unorthodox roles involve cell-cell interactions and are engaged to support immune functions such as leukocyte transmigration, recognition of opsonization factors, and stimulation of neutrophil extracellular traps. Other cell-cell interactions mediated by integrins include hematopoietic stem cell and tumor cell homing to target tissues. Integrins also serve as cell-surface receptors for various growth factors, hormones, and small molecules. Interestingly, integrins have also been exploited by a wide variety of organisms including viruses and bacteria to support infectious activities such as cellular adhesion and/or cellular internalization. Additionally, the disruption of integrin function through the use of soluble integrin ligands is a common strategy adopted by several parasites in order to inhibit blood clotting during hematophagy, or by venomous snakes to kill prey. In this review, we strive to go beyond the matrix and summarize non-ECM ligands that interact with integrins in order to highlight these non-traditional functions of integrins.
Smith, Julie A; Samayawardhena, Lionel A; Craig, Andrew W B
2010-03-01
Activation of Kit receptor protein-tyrosine kinase (PTK) by its ligand Stem Cell Factor (SCF) is required for the development of mast cells, and for the regulation of mast cell proliferation, migration and modulation of inflammatory mediator release. Recent studies have implicated the non-receptor PTK Fps/Fes (hereafter referred to as Fes) in signaling downstream of oncogenic Kit, however, the potential role of Fes in regulating Kit signaling is not well defined. In this study, we show that SCF induces transient tyrosine phosphorylation of wild-type Fes as well as kinase-dead Fes in bone marrow-derived mast cells (BMMCs). The latter finding implicates an upstream kinase acting on Fes, which we identified as Fyn PTK. SCF treatment of BMMCs promoted recruitment of Fes to Kit, potentially via direct interaction of the Fes SH2 domain with phosphorylated Kit. While Fes was not required for SCF-induced signaling to Akt and Erk kinases, Fes-deficient (fes-/-) BMMCs displayed a defect in sustained p38 kinase activation, compared to control cells. SCF-treated Fes-deficient BMMCs also displayed elevated beta1 integrin-mediated cell adhesion and spreading on fibronectin, compared to control cells, and a reduction in cell polarization at later times of SCF treatment. Restoring Fes expression in fes-/- BMMCs by retroviral transduction was sufficient to rescue cell spreading and polarization defects. Interestingly, SCF-induced chemotaxis of BMMCs was also defective in Fes-deficient BMMCs, and restored in Fes-rescue BMMCs. Overall, these results implicate Fes in regulating cross-talk between Kit and beta1 integrins to promote cytoskeletal reorganization and motility of mast cells.
Schmidt, Azriel; Vogel, Robert; Rutledge, Su Jane; Opas, Evan E; Rodan, Gideon A; Friedman, Eitan
2005-03-01
Nuclear receptors are transcription factors that usually interact, in a ligand-dependent manner, with specific DNA sequences located within promoters of target genes. The nuclear receptors can also be controlled in a ligand-independent manner via the action of membrane receptors and cellular signaling pathways. 5-Tetradecyloxy-2-furancarboxylic acid (TOFA) was shown to stimulate transcription from the MMTV promoter via chimeric receptors that consist of the DNA binding domain of GR and the ligand binding regions of the PPARbeta or LXRbeta nuclear receptors (GR/PPARbeta and GR/LXRbeta). TOFA and hydroxycholesterols also modulate transcription from NF-kappaB- and AP-1-controlled reporter genes and induce neurite differentiation in PC12 cells. In CV-1 cells that express D(1) dopamine receptors, D(1) dopamine receptor stimulation was found to inhibit TOFA-stimulated transcription from the MMTV promoter that is under the control of chimeric GR/PPARbeta and GR/LXRbeta receptors. Treatment with the D(1) dopamine receptor antagonist, SCH23390, prevented dopamine-mediated suppression of transcription, and by itself increased transcription controlled by GR/LXRbeta. Furthermore, combined treatment of CV-1 cells with TOFA and SCH23390 increased transcription controlled by the GR/LXRbeta chimeric receptor synergistically. The significance of this in vitro synergy was demonstrated in vivo, by the observation that SCH23390 (but not haloperidol)-mediated catalepsy in rats was potentiated by TOFA, thus showing that an agent that mimics the in vitro activities of compounds that activate members of the LXR and PPAR receptor families can influence D1 dopamine receptor elicited responses.
α4-integrin receptor desaturation and disease activity return after natalizumab cessation.
Derfuss, Tobias; Kovarik, John M; Kappos, Ludwig; Savelieva, Marina; Chhabra, Richa; Thakur, Avinash; Zhang, Ying; Wiendl, Heinz; Tomic, Davorka
2017-09-01
To describe the time course of α4-integrin receptor desaturation and disease activity return in patients with relapsing-remitting MS who discontinued natalizumab and to investigate baseline and on-study predictors for the recurrence of disease activity. In the course of TOFINGO, a 32-week, patient- and rater-blinded multicenter, parallel-group study, we performed MRI, counted relapses, and measured α4-integrin receptor occupancy (RO) at baseline and 8, 12, 16, 20, and 24 weeks. The relationship between RO and total number of new T1 gadolinium-enhancing (Gd+) lesions was modeled using Poisson linear regression. Patients (N = 142) were randomized (1:1:1) to 8-, 12-, or 16-week washout (WO) groups. At randomization, the median RO in the 8-, 12-, and 16-week WO groups was 94.5%, 92.4%, and 90.9%, which declined to 79.8%, 30.7%, and 8.7% after 8, 12, and 16 weeks of WO, respectively. The percentage of patients with new T1 Gd+ lesions increased with longer WO period before commencing fingolimod: 2.1% (8 weeks), 9.1% (12 weeks), and 50.0% (16 weeks). Overall, 71% of patients with first relapse between weeks 6 and 18 had RO values below the time-matched population median. Higher T2 lesion volume (LV) at baseline predicted a higher number of new T1 Gd+ lesions. A faster decline in natalizumab RO, longer WO period, and higher T2 LV at baseline were associated with an increased risk for return of inflammatory disease activity. These results provide a mechanistic rationale and, together with the main outcomes of the TOFINGO study, support initiation of fingolimod within 8 weeks of natalizumab discontinuation. NCT01499667.
Directory of Open Access Journals (Sweden)
Annika Sommerfeld
2015-05-01
Full Text Available Background/Aims: Ursodeoxycholic acid, which in vivo is rapidly converted into its taurine conjugate, is frequently used for the treatment of cholestatic liver disease. Apart from its choleretic effects, tauroursodeoxycholate (TUDC can protect hepatocytes from bile acid-induced apoptosis, but the mechanisms underlying its anti-apoptotic effects are poorly understood. Methods: These mechanisms were investigated in perfused rat liver and isolated rat hepatocytes. Results: It was found that TUDC inhibited the glycochenodeoxycholate (GCDC-induced activation of the CD95 death receptor at the level of association between CD95 and the epidermal growth factor receptor. This was due to a rapid TUDC-induced β1-integrin-dependent cyclic AMP (cAMP signal with induction of the dual specificity mitogen-activated protein (MAP kinase phosphatase 1 (MKP-1, which prevented GCDC-induced phosphorylation of mitogen-activated protein kinase kinase 4 (MKK4 and c-jun-NH2-terminal kinase (JNK activation. Furthermore, TUDC induced a protein kinase A (PKA-mediated serine/threonine phosphorylation of the CD95, which was recently identified as an internalization signal for CD95. Furthermore, TUDC inhibited GCDC-induced CD95 targeting to the plasma membrane in a β1-integrin-and PKA-dependent manner. In line with this, the β1-integrin siRNA knockdown in sodium taurocholate cotransporting polypeptide (Ntcp-transfected HepG2 cells abolished the protective effect of TUDC against GCDC-induced apoptosis. Conclusion: TUDC exerts its anti-apoptotic effect via a β1-integrin-mediated formation of cAMP, which prevents CD95 activation by hydrophobic bile acids at the levels of JNK activation and CD95 serine/threonine phosphorylation.
Duque, Hernando; Baxt, Barry
2003-01-01
Three members of the αV integrin family of cellular receptors, αVβ1, αVβ3, and αVβ6, have been identified as receptors for foot-and-mouth disease virus (FMDV) in vitro. The virus interacts with these receptors via a highly conserved arginine-glycine-aspartic acid (RGD) amino acid sequence motif located within the βG-βH (G-H) loop of VP1. Other αV integrins, as well as several other integrins, recognize and bind to RGD motifs on their natural ligands and also may be candidate receptors for FMDV. To analyze the roles of the αV integrins from a susceptible species as viral receptors, we molecularly cloned the bovine β1, β5, and β6 integrin subunits. Using these subunits, along with previously cloned bovine αV and β3 subunits, in a transient expression assay system, we compared the efficiencies of infection mediated by αVβ1, αVβ3, αVβ5, and αVβ6 among three strains of FMDV serotype A and two strains of serotype O. While all the viruses could infect cells expressing these integrins, they exhibited different efficiencies of integrin utilization. All the type A viruses used αVβ3 and αVβ6 with relatively high efficiency, while only one virus utilized αVβ1 with moderate efficiency. In contrast, both type O viruses utilized αVβ6 and αVβ1 with higher efficiency than αVβ3. Only low levels of viral replication were detected in αVβ5-expressing cells infected with either serotype. Experiments in which the ligand-binding domains among the β subunits were exchanged indicated that this region of the integrin subunit appears to contribute to the differences in integrin utilizations among strains. In contrast, the G-H loops of the different viruses do not appear to be involved in this phenomenon. Thus, the ability of the virus to utilize multiple integrins in vitro may be a reflection of the use of multiple receptors during the course of infection within the susceptible host. PMID:12551988
International Nuclear Information System (INIS)
Choudhuri, Tathagata; Verma, Subhash C.; Lan, Ke; Robertson, Erle S.
2006-01-01
Epstein-Barr virus (EBV) is a lymphotrophic herpesvirus infecting most of the world's population. It is associated with a number of human lymphoid and epithelial tumors and lymphoproliferative diseases in immunocompromised patients. A subset of latent EBV antigens is required for immortalization of primary B-lymphocytes. The metastatic suppressor Nm23-H1 which is downregulated in human invasive breast carcinoma reduces the migration and metastatic activity of breast carcinoma cells when expressed from a heterologous promoter. Interestingly, the EBV nuclear antigen 3C (EBNA3C) reverses these activities of Nm23-H1. The alpha V integrins recognize a variety of ligands for signaling and are involved in cell migration and proliferation and also serve as major receptors for extracellular-matrix-mediated cell adhesion and migration. The goal of this study was to determine if Nm23-H1 and EBNA3C can modulate alpha V integrin expression and downstream activities. The results of our studies indicate that Nm23-H1 downregulates alpha V intregrin expression in a dose responsive manner. In contrast, EBNA3C can upregulate alpha V integrin expression. Furthermore, the study showed that the association of the Sp1 and GATA transcription factors with Nm23-H1 is required for modulation of the alpha V integrin activity. Thus, these results suggest a direct correlation between the alpha V integrin expression and the interaction of Nm23-H1 with EBNA3C
Schlaepfer, D D; Hanks, S K; Hunter, T; van der Geer, P
The cytoplasmic focal adhesion protein-tyrosine kinase (FAK) localizes with surface integrin receptors at sites where cells attach to the extracellular matrix. Increased FAK tyrosine phosphorylation occurs upon integrin engagement with fibronectin. Here we show that adhesion of murine NIH3T3 fibroblasts to fibronectin promotes SH2-domain-mediated association of the GRB2 adaptor protein and the c-Src protein-tyrosine kinase (PTK) with FAK in vivo, and also results in activation of mitogen-activated protein kinase (MAPK). In v-Src-transformed NIH3T3, the association of v-Src, GRB2 and Sos with FAK is independent of cell adhesion to fibronectin. The GRB2 SH2 domain binds directly to tyrosine-phosphorylated FAK. Mutation of tyrosine residue 925 of FAK (YENV motif) to phenylalanine blocks GRB2 SH2-domain binding to FAK in vitro. Our results show that fibronectin binding to integrins on NIH3T3 fibroblasts promotes c-Src and FAK association and formation of an integrin-activated signalling complex. Phosphorylation of FAK at Tyr 925 upon fibronectin stimulation creates an SH2-binding site for GRB2 which may link integrin engagement to the activation of the Ras/MAPK signal transduction pathway.
Ofloxacin induces apoptosis via β1 integrin-EGFR-Rac1-Nox2 pathway in microencapsulated chondrocytes
International Nuclear Information System (INIS)
Sheng, Zhi-Guo; Huang, Wei; Liu, Yu-Xiang; Yuan, Ye; Zhu, Ben-Zhan
2013-01-01
Quinolones (QNs)-induced arthropathy is an important toxic side-effect in immature animals leading to the restriction of their therapeutic use in pediatrics. Ofloxacin, a typical QN, was found to induce the chondrocytes apoptosis in the early phase (12–48 h) of arthropathy in our previous study. However, the exact mechanism(s) is unclear. Microencapsulated juvenile rabbit joint chondrocytes, a three-dimensional culture system, is utilized to perform the present study. Ofloxacin, at a therapeutically relevant concentration (10 μg/ml), disturbs the interaction between β1 integrin and activated intracellular signaling proteins at 12 h, which is inhibited when supplementing Mg 2+ . Intracellular reactive oxygen species (ROS) significantly increases in a time-dependent manner after exposure to ofloxacin for 12–48 h. Furthermore, ofloxacin markedly enhances the level of activated Rac1 and epidermal growth factor receptor (EGFR) phosphorylation, and its inhibition in turn reduces the ROS production, apoptosis and Rac1 activation. Silencing Nox2, Rac1 or supplementing Mg 2+ inhibits ROS accumulation, apoptosis occurrence and EGFR phosphorylation induced by ofloxacin. However, depletion of Nox2, Rac1 and inhibition of EGFR do not affect ofloxacin-mediated loss of interaction between β1 integrin and activated intracellular signaling proteins. In addition, ofloxacin also induces Vav2 phosphorylation, which is markedly suppressed after inactivating EGFR or supplementing Mg 2+ . These results suggest that ofloxacin causes Nox2-mediated intracellular ROS production by disrupting the β1 integrin function and then activating the EGFR-Vav2-Rac1 pathway, finally resulting in apoptosis within 12–48 h exposure. The present study provides a novel insight regarding the potential role of Nox-driven ROS in QNs-induced arthropathy. - Highlights: ► Ofloxacin induces Nox2-driven ROS in encapsulated chondrocyte at 12–48 h. ► Ofloxacin stimulates ROS production via the β1
Ofloxacin induces apoptosis via β1 integrin-EGFR-Rac1-Nox2 pathway in microencapsulated chondrocytes
Energy Technology Data Exchange (ETDEWEB)
Sheng, Zhi-Guo [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Science, Chinese Academy of Sciences, 18 Shuangqing Road, Beijing 100085 (China); Huang, Wei [Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 1000191 (China); Liu, Yu-Xiang [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Science, Chinese Academy of Sciences, 18 Shuangqing Road, Beijing 100085 (China); Yuan, Ye [Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Beijing 100850 (China); Zhu, Ben-Zhan, E-mail: bzhu@rcees.ac.cn [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Science, Chinese Academy of Sciences, 18 Shuangqing Road, Beijing 100085 (China); Linus Pauling Institute, Oregon State University, Corvallis, OR 97331 (United States)
2013-02-15
Quinolones (QNs)-induced arthropathy is an important toxic side-effect in immature animals leading to the restriction of their therapeutic use in pediatrics. Ofloxacin, a typical QN, was found to induce the chondrocytes apoptosis in the early phase (12–48 h) of arthropathy in our previous study. However, the exact mechanism(s) is unclear. Microencapsulated juvenile rabbit joint chondrocytes, a three-dimensional culture system, is utilized to perform the present study. Ofloxacin, at a therapeutically relevant concentration (10 μg/ml), disturbs the interaction between β1 integrin and activated intracellular signaling proteins at 12 h, which is inhibited when supplementing Mg{sup 2+}. Intracellular reactive oxygen species (ROS) significantly increases in a time-dependent manner after exposure to ofloxacin for 12–48 h. Furthermore, ofloxacin markedly enhances the level of activated Rac1 and epidermal growth factor receptor (EGFR) phosphorylation, and its inhibition in turn reduces the ROS production, apoptosis and Rac1 activation. Silencing Nox2, Rac1 or supplementing Mg{sup 2+} inhibits ROS accumulation, apoptosis occurrence and EGFR phosphorylation induced by ofloxacin. However, depletion of Nox2, Rac1 and inhibition of EGFR do not affect ofloxacin-mediated loss of interaction between β1 integrin and activated intracellular signaling proteins. In addition, ofloxacin also induces Vav2 phosphorylation, which is markedly suppressed after inactivating EGFR or supplementing Mg{sup 2+}. These results suggest that ofloxacin causes Nox2-mediated intracellular ROS production by disrupting the β1 integrin function and then activating the EGFR-Vav2-Rac1 pathway, finally resulting in apoptosis within 12–48 h exposure. The present study provides a novel insight regarding the potential role of Nox-driven ROS in QNs-induced arthropathy. - Highlights: ► Ofloxacin induces Nox2-driven ROS in encapsulated chondrocyte at 12–48 h. ► Ofloxacin stimulates ROS production via
Sarathy, Apurva; Wuebbles, Ryan D; Fontelonga, Tatiana M; Tarchione, Ashley R; Mathews Griner, Lesley A; Heredia, Dante J; Nunes, Andreia M; Duan, Suzann; Brewer, Paul D; Van Ry, Tyler; Hennig, Grant W; Gould, Thomas W; Dulcey, Andrés E; Wang, Amy; Xu, Xin; Chen, Catherine Z; Hu, Xin; Zheng, Wei; Southall, Noel; Ferrer, Marc; Marugan, Juan; Burkin, Dean J
2017-06-07
Duchenne muscular dystrophy (DMD) is a fatal muscle disease caused by mutations in the dystrophin gene, resulting in a complete loss of the dystrophin protein. Dystrophin is a critical component of the dystrophin glycoprotein complex (DGC), which links laminin in the extracellular matrix to the actin cytoskeleton within myofibers and provides resistance to shear stresses during muscle activity. Loss of dystrophin in DMD patients results in a fragile sarcolemma prone to contraction-induced muscle damage. The α7β1 integrin is a laminin receptor protein complex in skeletal and cardiac muscle and a major modifier of disease progression in DMD. In a muscle cell-based screen for α7 integrin transcriptional enhancers, we identified a small molecule, SU9516, that promoted increased α7β1 integrin expression. Here we show that SU9516 leads to increased α7B integrin in murine C2C12 and human DMD patient myogenic cell lines. Oral administration of SU9516 in the mdx mouse model of DMD increased α7β1 integrin in skeletal muscle, ameliorated pathology, and improved muscle function. We show that these improvements are mediated through SU9516 inhibitory actions on the p65-NF-κB pro-inflammatory and Ste20-related proline alanine rich kinase (SPAK)/OSR1 signaling pathways. This study identifies a first in-class α7 integrin-enhancing small-molecule compound with potential for the treatment of DMD. Copyright © 2017 The American Society of Gene and Cell Therapy. All rights reserved.
Vascular endothelin ET(B) receptor-mediated contraction requires phosphorylation of ERK1/2 proteins
DEFF Research Database (Denmark)
Luo, Guogang; Jamali, Roya; Cao, Yong-Xiao
2006-01-01
In cardiovascular diseases, endothelin type B (ET(B)) receptors in arterial smooth muscle cells are upregulated. The present study revealed that organ culture of rat mesenteric artery segments enhanced endothelin ET(B) receptor-mediated contraction paralleled with increase in the receptor mRNA an...
The Interaction of CD154 with the α5β1 Integrin Inhibits Fas-Induced T Cell Death.
Bachsais, Meriem; Naddaf, Nadim; Yacoub, Daniel; Salti, Suzanne; Alaaeddine, Nada; Aoudjit, Fawzi; Hassan, Ghada S; Mourad, Walid
2016-01-01
CD154, a critical regulator of the immune response, is usually associated with chronic inflammatory, autoimmune diseases as well as malignant disorders. In addition to its classical receptor CD40, CD154 is capable of binding other receptors, members of the integrin family, the αIIbβ3, αMβ2 and α5β1. Given the role attributed to integrins and particularly the β1 integrins in inhibiting apoptotic events in normal as well as malignant T cells, we were highly interested in investigating the role of the CD154/α5β1 interaction in promoting survival of malignant T cells contributing as such to tumor development and/or propagation. To support our hypothesis, we first show that soluble CD154 binds to the T-cell acute lymphoblastic leukemia cell line, Jurkat E6.1 in a α5β1-dependent manner. Binding of soluble CD154 to α5β1 integrin of Jurkat cells leads to the activation of key survival proteins, including the p38 and ERK1/2 mitogen-activated protein kinases (MAPKs), phosphoinositide 3 kinase (PI-3K), and Akt. Interestingly, soluble CD154 significantly inhibits Fas-mediated apoptosis in T cell leukemia-lymphoma cell lines, Jurkat E6.1 and HUT78 cells, an important hallmark of T cell survival during malignancy progression. These anti-apoptotic effects were mainly mediated by the activation of the PI-3K/Akt pathway but also involved the p38 and the ERK1/2 MAPKs cascades. Our data also demonstrated that the CD154-triggered inhibition of the Fas-mediated cell death response was dependent on a suppression of caspase-8 cleavage, but independent of de novo protein synthesis or alterations in Fas expression on cell surface. Together, our results highlight the impact of the CD154/α5β1 interaction in T cell function/survival and identify novel targets for the treatment of malignant disorders, particularly of T cell origin.
The Interaction of CD154 with the α5β1 Integrin Inhibits Fas-Induced T Cell Death.
Directory of Open Access Journals (Sweden)
Meriem Bachsais
Full Text Available CD154, a critical regulator of the immune response, is usually associated with chronic inflammatory, autoimmune diseases as well as malignant disorders. In addition to its classical receptor CD40, CD154 is capable of binding other receptors, members of the integrin family, the αIIbβ3, αMβ2 and α5β1. Given the role attributed to integrins and particularly the β1 integrins in inhibiting apoptotic events in normal as well as malignant T cells, we were highly interested in investigating the role of the CD154/α5β1 interaction in promoting survival of malignant T cells contributing as such to tumor development and/or propagation. To support our hypothesis, we first show that soluble CD154 binds to the T-cell acute lymphoblastic leukemia cell line, Jurkat E6.1 in a α5β1-dependent manner. Binding of soluble CD154 to α5β1 integrin of Jurkat cells leads to the activation of key survival proteins, including the p38 and ERK1/2 mitogen-activated protein kinases (MAPKs, phosphoinositide 3 kinase (PI-3K, and Akt. Interestingly, soluble CD154 significantly inhibits Fas-mediated apoptosis in T cell leukemia-lymphoma cell lines, Jurkat E6.1 and HUT78 cells, an important hallmark of T cell survival during malignancy progression. These anti-apoptotic effects were mainly mediated by the activation of the PI-3K/Akt pathway but also involved the p38 and the ERK1/2 MAPKs cascades. Our data also demonstrated that the CD154-triggered inhibition of the Fas-mediated cell death response was dependent on a suppression of caspase-8 cleavage, but independent of de novo protein synthesis or alterations in Fas expression on cell surface. Together, our results highlight the impact of the CD154/α5β1 interaction in T cell function/survival and identify novel targets for the treatment of malignant disorders, particularly of T cell origin.
Directory of Open Access Journals (Sweden)
Elena Rainero
Full Text Available Diacylglycerol kinase α (DGKα, by phosphorylating diacylglycerol into phosphatidic acid, provides a key signal driving cell migration and matrix invasion. We previously demonstrated that in epithelial cells activation of DGKα activity promotes cytoskeletal remodeling and matrix invasion by recruiting atypical PKC at ruffling sites and by promoting RCP-mediated recycling of α5β1 integrin to the tip of pseudopods. In here we investigate the signaling pathway by which DGKα mediates SDF-1α-induced matrix invasion of MDA-MB-231 invasive breast carcinoma cells. Indeed we showed that, following SDF-1α stimulation, DGKα is activated and localized at cell protrusion, thus promoting their elongation and mediating SDF-1α induced MMP-9 metalloproteinase secretion and matrix invasion. Phosphatidic acid generated by DGKα promotes localization at cell protrusions of atypical PKCs which play an essential role downstream of DGKα by promoting Rac-mediated protrusion elongation and localized recruitment of β1 integrin and MMP-9. We finally demonstrate that activation of DGKα, atypical PKCs signaling and β1 integrin are all essential for MDA-MB-231 invasiveness. These data indicates the existence of a SDF-1α induced DGKα - atypical PKC - β1 integrin signaling pathway, which is essential for matrix invasion of carcinoma cells.
DEFF Research Database (Denmark)
Brevig, T.; Holst, B.; Ademovic, Z.
2005-01-01
Leukocyte beta(2) integrins Mac-1 and p150,95 are promiscuous cell-surface receptors that recognise and mediate cell adhesion to a variety of adsorbed and denatured proteins. We used albumin as a model protein to study whether leukocyte adhesion and activation depended on the nm-scale topography...
In vitro binding and receptor-mediated activity of terlipressin at vasopressin receptors V1 and V2.
Jamil, Khurram; Pappas, Stephen Chris; Devarakonda, Krishna R
2018-01-01
Terlipressin, a synthetic, systemic vasoconstrictor with selective activity at vasopressin-1 (V 1 ) receptors, is a pro-drug for the endogenous/natural porcine hormone [Lys 8 ]-vasopressin (LVP). We investigated binding and receptor-mediated cellular activities of terlipressin, LVP, and endogenous human hormone [Arg 8 ]-vasopressin (AVP) at V 1 and vasopressin-2 (V 2 ) receptors. Cell membrane homogenates of Chinese hamster ovary cells expressing human V 1 and V 2 receptors were used in competitive binding assays to measure receptor-binding activity. These cells were used in functional assays to measure receptor-mediated cellular activity of terlipressin, LVP, and AVP. Binding was measured by [ 3 H]AVP counts, and the activity was measured by fluorometric detection of intracellular calcium mobilization (V 1 ) and cyclic adenosine monophosphate (V 2 ). Binding potency at V 1 and V 2 was AVP>LVP>terlipressin. LVP and terlipressin had approximately sixfold higher affinity for V 1 than for V 2 . Cellular activity potency was also AVP>LVP>terlipressin. Terlipressin was a partial agonist at V 1 and a full agonist at V 2 ; LVP was a full agonist at both V 1 and V 2 . The in vivo response to terlipressin is likely due to the partial V 1 agonist activity of terlipressin and full V 1 agonist activity of its metabolite, LVP. These results provide supportive evidence for previous findings and further establish terlipressin pharmacology for vasopressin receptors.
Directory of Open Access Journals (Sweden)
Carlos E Caicedo-Carvajal
2010-07-01
Full Text Available Tissue organization during embryonic development and wound healing depends on the ability of cells on the one hand to exchange adhesive bonds during active rearrangement and on the other to become fixed in place as tissue homeostasis is reached. Cells achieve these contradictory tasks by regulating either cell-cell adhesive bonds, mediated by cadherins, or cell-extracellular matrix (ECM connections, regulated by integrins. Integrin alpha5beta1 and soluble fibronectin (sFN are key players in cell-ECM force generation and in ECM polymerization. Here, we explore the interplay between integrin alpha5beta1 and sFN and its influence on tissue mechanical properties and cell sorting behavior.We generated a series of cell lines varying in alpha5beta1 receptor density. We then systematically explored the effects of different sFN concentrations on aggregate biomechanical properties using tissue surface tensiometry. We found previously unreported complex behaviors including the observation that interactions between fibronectin and integrin alpha5beta1 generates biphasic tissue cohesion profiles. Specifically, we show that at constant sFn concentration, aggregate cohesion increases linearly as alpha5beta1 receptor density is increased from low to moderate levels, producing a transition from viscoelastic-liquid to pseudo viscoelastic-solid behavior. However, further increase in receptor density causes an abrupt drop in tissue cohesion and a transition back to viscoelastic-liquid properties. We propose that this may be due to depletion of sFn below a critical value in the aggregate microenvironment at high alpha5beta1 levels. We also show that differential expression of alpha5beta1 integrin can promote phase-separation between cells.The interplay between alpha5-integrin and sFn contributes significantly to tissue cohesion and, depending on their level of expression, can mediate a shift from liquid to elastic behavior. This interplay represents a tunable level
Scavenger receptor B1 facilitates macrophage uptake of silver nanoparticles and cellular activation
Energy Technology Data Exchange (ETDEWEB)
Aldossari, Abdullah A.; Shannahan, Jonathan H. [The University of Colorado Anschutz Medical Campus, Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences (United States); Podila, Ramakrishna [Clemson University, Department of Physics and Astronomy (United States); Brown, Jared M., E-mail: jared.brown@ucdenver.edu [The University of Colorado Anschutz Medical Campus, Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences (United States)
2015-07-15
Due to increased use of silver nanoparticles (AgNPs) for their antimicrobial activity, concerns have risen regarding potential adverse human health effects. Scavenger receptor B1 (SR-B1), a major receptor for high-density lipoprotein (HDL), is expressed by macrophages and has also been reported to play a role in recognition of negatively charged particles. We, therefore, hypothesized that SR-B1 mediates macrophage uptake of AgNPs and inflammatory activation. To test this hypothesis, we exposed a mouse macrophage cell line RAW264.7 (RAW) and bone marrow-derived macrophages (BMDM) to 20 nm citrate-suspended AgNPs. To verify the role of the SR-B1 receptor, we utilized a SR-B1 inhibitor (Blt2). In vitro studies demonstrated uptake of AgNPs and HDL-coated AgNPs by macrophages which were significantly reduced following pretreatment with Blt2. Inflammatory cytokine arrays revealed that macrophages exposed to AgNPs up-regulated expression of Tnf-α, Oncostatin m (OSM), Ccl4, Il17f, Ccl7, and Ccl2, whereas Il16 was found to be down-regulated. Macrophage activation was observed following AgNP and HDL-coated AgNP exposure as measured by OSM protein production and increased surface expression of CD86. These markers of activation were reduced with Blt2 pretreatment. The in vitro findings were confirmed in vivo through pulmonary instillation of AgNPs in mice. Pulmonary instillation of AgNPs resulted in a recruitment of inflammatory cells that were reduced in SR-B1-deficient mice or following Blt2 pretreatment. This study suggests that SR-B1 plays a major role in cellular recognition of AgNPs and the induction of cell responses that could contribute to inflammation caused by AgNP exposure.
Scavenger receptor B1 facilitates macrophage uptake of silver nanoparticles and cellular activation
Aldossari, Abdullah A.; Shannahan, Jonathan H.; Podila, Ramakrishna; Brown, Jared M.
2015-07-01
Due to increased use of silver nanoparticles (AgNPs) for their antimicrobial activity, concerns have risen regarding potential adverse human health effects. Scavenger receptor B1 (SR-B1), a major receptor for high-density lipoprotein (HDL), is expressed by macrophages and has also been reported to play a role in recognition of negatively charged particles. We, therefore, hypothesized that SR-B1 mediates macrophage uptake of AgNPs and inflammatory activation. To test this hypothesis, we exposed a mouse macrophage cell line RAW264.7 (RAW) and bone marrow-derived macrophages (BMDM) to 20 nm citrate-suspended AgNPs. To verify the role of the SR-B1 receptor, we utilized a SR-B1 inhibitor (Blt2). In vitro studies demonstrated uptake of AgNPs and HDL-coated AgNPs by macrophages which were significantly reduced following pretreatment with Blt2. Inflammatory cytokine arrays revealed that macrophages exposed to AgNPs up-regulated expression of Tnf- α, Oncostatin m (OSM), Ccl4, Il17f, Ccl7, and Ccl2, whereas Il16 was found to be down-regulated. Macrophage activation was observed following AgNP and HDL-coated AgNP exposure as measured by OSM protein production and increased surface expression of CD86. These markers of activation were reduced with Blt2 pretreatment. The in vitro findings were confirmed in vivo through pulmonary instillation of AgNPs in mice. Pulmonary instillation of AgNPs resulted in a recruitment of inflammatory cells that were reduced in SR-B1-deficient mice or following Blt2 pretreatment. This study suggests that SR-B1 plays a major role in cellular recognition of AgNPs and the induction of cell responses that could contribute to inflammation caused by AgNP exposure.
Cohn, R D; Mayer, U; Saher, G; Herrmann, R; van der Flier, A; Sonnenberg, A; Sorokin, L; Voit, T
1999-03-01
The integrins are a large family of heterodimeric transmembrane cellular receptors which mediate the association between the extracellular matrix (ECM) and cytoskeletal proteins. The alpha7beta1 integrin is a major laminin binding integrin in skeletal and cardiac muscle and is thought to be involved in myogenic differentiation and migration processes. The main binding partners of the alpha7 integrin are laminin-1 (alpha1-beta1-gamma1), laminin-2 (alpha2-beta1-gamma1) and laminin-4 (alpha2-beta2-gamma1). Targeted deletion of the gene for the alpha7 integrin subunit (ITGA7) in mice leads to a novel form of muscular dystrophy. In the present study we have investigated the expression of two alternative splice variants, the alpha7B and beta1D integrin subunits, in normal human skeletal muscle, as well as in various forms of muscular dystrophy. In normal human skeletal muscle the expression of the alpha7 integrin subunit appeared to be developmentally regulated: it was first detected at 2 years of age. In contrast, the beta1D integrin could be detected in immature and mature muscle in the sarcolemma of normal fetal skeletal muscle at 18 weeks gestation. The expression of alpha7B integrin was significantly reduced at the sarcolemma in six patients with laminin alpha2 chain deficient congenital muscular dystrophy (CMD) (age >2 years). However, this reduction was not correlated with the amount of laminin alpha2 chain expressed. In contrast, the expression of the laminin alpha2 chain was not altered in the skeletal muscle of the alpha7 knock-out mice. These data argue in favor that there is not a tight correlation between the expression of the alpha7 integrin subunit and that of the laminin alpha2 chain in either human or murine dystrophic muscle. Interestingly, in dystrophinopathies (Duchenne and Becker muscular dystrophy; DMD/BMD) expression of alpha7B was upregulated irrespective of the level of dystrophin expression as shown by a strong sarcolemmal staining pattern even
Role of β1 Integrin in Tissue Homing of Neutrophils During Sepsis
Sarangi, Pranita P.; Hyun, Young-Min; Lerman, Yelena V.; Pietropaoli, Anthony P.; Kim, Minsoo
2012-01-01
Aberrant activation of neutrophils during sepsis results in the widespread release of pro-inflammatory mediators, leading to multi-organ system failure and death. However, aberrant activation of neutrophils during sepsis results in the widespread release of harmful inflammatory mediators causing host tissue injuries that can lead to multi organ system failure and death. One of the pivotal components of neutrophil migration during inflammation is the expression of surface integrins. In this study, we show that administration of a cyclic analog of RGD peptide (Arg-Gly-Asp) significantly reduced the number of tissue-invading neutrophils and the degree of sepsis-induced lethality in mice as compared to control peptide. Secondly, β1 integrin (CD29) was highly up-regulated on the neutrophils isolated from both septic patients and animals. Finally, conditional genetic ablation of β1 integrin from granulocytes also improved survival and bacterial clearance in septic animals Thus, our results indicate that expression of β1 integrin is important for modulating neutrophil trafficking during sepsis, and that therapeutics designed against β1 integrins may be beneficial. PMID:22683734
Directory of Open Access Journals (Sweden)
Nilgun Gurbuz
Full Text Available Pancreatic ductal adenocarcinoma is characterized by extensive local tumor invasion, metastasis and early systemic dissemination. The vast majority of pancreatic cancer (PaCa patients already have metastatic complications at the time of diagnosis, and the death rate of this lethal type of cancer has increased over the past decades. Thus, efforts at identifying novel molecularly targeted therapies are priorities. Recent studies have suggested that serotonin (5-HT contributes to the tumor growth in a variety of cancers including prostate, colon, bladder and liver cancer. However, there is lack of evidence about the impact of 5-HT receptors on promoting pancreatic cancer. Having considered the role of 5-HT-1 receptors, especially 5-HT1B and 5-HT1D subtypes in different types of malignancies, the aim of this study was to investigate the role of 5-HT1B and 5-HT1D receptors in PaCa growth and progression and analyze their potential as cytotoxic targets. We found that knockdown of 5-HT1B and 5-HT1D receptors expression, using specific small interfering RNA (siRNA, induced significant inhibition of proliferation and clonogenicity of PaCa cells. Also, it significantly suppressed PaCa cells invasion and reduced the activity of uPAR/MMP-2 signaling and Integrin/Src/Fak-mediated signaling, as integral tumor cell pathways associated with invasion, migration, adhesion, and proliferation. Moreover, targeting 5-HT1B and 5-HT1D receptors down-regulates zinc finger ZEB1 and Snail proteins, the hallmarks transcription factors regulating epithelial-mesenchymal transition (EMT, concomitantly with up-regulating of claudin-1 and E-Cadherin. In conclusion, our data suggests that 5-HT1B- and 5-HT1D-mediated signaling play an important role in the regulation of the proliferative and invasive phenotype of PaCa. It also highlights the therapeutic potential of targeting of 5-HT1B/1D receptors in the treatment of PaCa, and opens a new avenue for biomarkers identification
Directory of Open Access Journals (Sweden)
Nilgun Gurbuz
Full Text Available Pancreatic ductal adenocarcinoma is characterized by extensive local tumor invasion, metastasis and early systemic dissemination. The vast majority of pancreatic cancer (PaCa patients already have metastatic complications at the time of diagnosis, and the death rate of this lethal type of cancer has increased over the past decades. Thus, efforts at identifying novel molecularly targeted therapies are priorities. Recent studies have suggested that serotonin (5-HT contributes to the tumor growth in a variety of cancers including prostate, colon, bladder and liver cancer. However, there is lack of evidence about the impact of 5-HT receptors on promoting pancreatic cancer. Having considered the role of 5-HT-1 receptors, especially 5-HT1B and 5-HT1D subtypes in different types of malignancies, the aim of this study was to investigate the role of 5-HT1B and 5-HT1D receptors in PaCa growth and progression and analyze their potential as cytotoxic targets. We found that knockdown of 5-HT1B and 5-HT1D receptors expression, using specific small interfering RNA (siRNA, induced significant inhibition of proliferation and clonogenicity of PaCa cells. Also, it significantly suppressed PaCa cells invasion and reduced the activity of uPAR/MMP-2 signaling and Integrin/Src/Fak-mediated signaling, as integral tumor cell pathways associated with invasion, migration, adhesion, and proliferation. Moreover, targeting 5-HT1B and 5-HT1D receptors down-regulates zinc finger ZEB1 and Snail proteins, the hallmarks transcription factors regulating epithelial-mesenchymal transition (EMT, concomitantly with up-regulating of claudin-1 and E-Cadherin. In conclusion, our data suggests that 5-HT1B- and 5-HT1D- mediated signaling play an important role in the regulation of the proliferative and invasive phenotype of PaCa. It also highlights the therapeutic potential of targeting of 5-HT1B/1D receptors in the treatment of PaCa, and opens a new avenue for biomarkers identification
DEFF Research Database (Denmark)
Underwood, Christina Rye; Møller Knudsen, Sanne; Schjellerup Wulff, Birgitte
2011-01-01
Glucagon-like peptide-1 (GLP-1) activates the GLP-1 receptor (GLP-1R), which belongs to family B of the G-protein-coupled receptors. We previously identified a selective small molecule ligand, compound 2, that acted as a full agonist and allosteric modulator of GLP-1R. In this study, the structur......Glucagon-like peptide-1 (GLP-1) activates the GLP-1 receptor (GLP-1R), which belongs to family B of the G-protein-coupled receptors. We previously identified a selective small molecule ligand, compound 2, that acted as a full agonist and allosteric modulator of GLP-1R. In this study......, the structurally related small molecule, compound 3, stimulated cAMP production from GLP-1R, but not from the homologous glucagon receptor (GluR). The receptor selectivity encouraged a chimeric receptor approach to identify domains important for compound 3-mediated activation of GLP-1R. A subsegment of the GLP-1R...... transmembrane domain containing TM2 to TM5 was sufficient to transfer compound 3 responsiveness to GluR. Therefore, divergent residues in this subsegment of GLP-1R and GluR are responsible for the receptor selectivity of compound 3. Functional analyses of other chimeric receptors suggested that the existence...
The involvement of Gab1 and PI 3-kinase in β1 integrin signaling in keratinocytes
International Nuclear Information System (INIS)
Kuwano, Yoshihiro; Fujimoto, Manabu; Watanabe, Rei; Ishiura, Nobuko; Nakashima, Hiroko; Komine, Mayumi; Hamazaki, Tatsuo S.; Tamaki, Kunihiko; Okochi, Hitoshi
2007-01-01
The control of the stem cell compartment in epidermis is closely linked to the regulation of keratinocyte proliferation and differentiation. β1 integrins are expressed 2-fold higher by stem cells than transit-amplifying cells. Signaling from these β1 integrins is critical for the regulation of the epidermal stem cell compartment. To clarify the functional relevance of this differential expression of β1 integrins, we established HaCaT cells with high β1integrin expression by repeated flow cytometric sorting of this population from the parental cell line. In these obtained cells expressing β1 integrins by 5-fold, MAPK activation was markedly increased. Regarding the upstream of MAPK, Gab1 phosphorylation was also higher with high β1 integrin expression, while Shc phosphorylation was not altered. In addition, enhanced phosphatidylinositol 3-kinase activation was also observed. These observations suggest that Gab1 and phosphatidylinositol 3-kinase play pivotal roles in the β1 integrin-mediated regulation of the epidermal stem cell compartment
Schelfaut, Roselien
2005-01-01
Integrins are receptors presented on most cells. By binding ligand they can generate signalling pathways inside the cell. Those pathways are a linkage to proteins in the cytosol. It is known that tumor cells can survive and proliferate in the absence of a solid support while normal cells need to be bound to ligand. To understand why tumour cells act that way, we first have to know how ligand-binding to integrins affect the cell. This research field includes studies on activation of proteins b...
Directory of Open Access Journals (Sweden)
Selistre-de-Araujo H.S.
2005-01-01
Full Text Available The alpha2ß1 integrin is a major collagen receptor that plays an essential role in the adhesion of normal and tumor cells to the extracellular matrix. Alternagin-C (ALT-C, a disintegrin-like protein purified from the venom of the Brazilian snake Bothrops alternatus, competitively interacts with the alpha2ß1 integrin, thereby inhibiting collagen binding. When immobilized in plate wells, ALT-C supports the adhesion of fibroblasts as well as of human vein endothelial cells (HUVEC and does not detach cells previously bound to collagen I. ALT-C is a strong inducer of HUVEC proliferation in vitro. Gene expression analysis was done using an Affimetrix HU-95A probe array with probe sets of ~10,000 human genes. In human fibroblasts growing on collagen-coated plates, ALT-C up-regulates the expression of several growth factors including vascular endothelial growth factor, as well as some cell cycle control genes. Up-regulation of the vascular endothelial growth factor gene and other growth factors could explain the positive effect on HUVEC proliferation. ALT-C also strongly activates protein kinase B phosphorylation, a signaling event involved in endothelial cell survival and angiogenesis. In human neutrophils, ALT-C has a potent chemotactic effect modulated by the intracellular signaling cascade characteristic of integrin-activated pathways. Thus, ALT-C acts as a survival factor, promoting adhesion, migration and endothelial cell proliferation after binding to alpha2ß1 integrin on the cell surface. The biological activities of ALT-C may be helpful as a therapeutic strategy in tissue regeneration as well as in the design of new therapeutic agents targeting alpha2ß1 integrin.
International Nuclear Information System (INIS)
Hollenbeck, Scott T.; Itoh, Hiroyuki; Louie, Otway; Faries, Peter L.; Liu Bo; Kent, K. Craig
2004-01-01
Smooth muscle cells (SMCs) are exposed to both platelet-derived growth factor (PDGF) and type I collagen (CNI) at the time of arterial injury. In these studies we explore the individual and combined effects of these agonists on human saphenous vein SMC proliferation. PDGF-BB produced a 5.5-fold increase in SMC DNA synthesis whereas CNI stimulated DNA synthesis to a much lesser extent (1.6-fold increase). Alternatively, we observed an 8.3-fold increase in DNA synthesis when SMCs were co-incubated with CNI and PDGF-BB. Furthermore, stimulation of SMCs with PDGF-BB produced a significant increase in ERK-2 activity whereas CNI alone had no effect. Co-incubation of SMCs with PDGF-BB and CNI resulted in ERK-2 activity that was markedly greater than that produced by PDGF-BB alone. In a similar fashion, PDGF-BB induced phosphorylation of the PDGF receptor β (PDGFRβ) and CNI did not, whereas concurrent agonist stimulation produced a synergistic increase in receptor activity. Blocking antibodies to the α2 and β1 subunits eliminated this synergistic interaction, implicating the α2β1 integrin as the mediator of this effect. Immunoprecipitation of the α2β1 integrin in unstimulated SMCs followed by immunoblotting for the PDGFRβ as well as Src family members, pp60 src , Fyn, Lyn, and Yes demonstrated coassociation of α2β1 and the PDGFRβ as well as pp60 src . Incubation of cells with CNI and/or PDGF-BB did not change the degree of association. Finally, inhibition of Src activity with SU6656 eliminated the synergistic effect of CNI on PDGF-induced PDGFRβ phosphorylation suggesting an important role for pp60 src in the observed receptor crosstalk. Together, these data demonstrate that CNI synergistically enhances PDGF-induced SMC proliferation through Src-dependent crosstalk between the α2β1 integrin and the PDGFRβ
Ulfman, L.H.; Kuijper, P.H.M.; Linden, J.A.M. van der; Lammers, J.W.J.; Zwaginga, Jaap Jan; Koenderman, L.
1999-01-01
The multistep model of leukocyte adhesion reveals that selectins mediate rolling interactions and that integrins mediate firm adhesion processes. In this study, the interaction between eosinophils and TNF-a-activated HUVEC (second or third passage) was studied under flow conditions (0.8 and 3.2
Activation of c-Raf-1 kinase signal transduction pathway in alpha(7) integrin-deficient mice.
Saher, G; Hildt, E
1999-09-24
Integrin alpha(7)-deficient mice develop a novel form of muscular dystrophy. Here we report that deficiency of alpha(7) integrin causes an activation of the c-Raf-1/mitogen-activated protein (MAP) 2 kinase signal transduction pathway in muscle cells. The observed activation of c-Raf-1/MAP2 kinases is a specific effect, because the alpha(7) integrin deficiency does not cause unspecific stress as determined by measurement of the Hsp72/73 level and activity of the JNK2 kinase. Because an increased level of activated FAK was found in muscle of alpha(7) integrin-deficient mice, the activation of c-Raf-1 kinase is triggered most likely by an integrin-dependent pathway. In accordance with this, in the integrin alpha(7)-deficient mice, part of the integrin beta(1D) variant in muscle is replaced by the beta(1A) variant, which permits the FAK activation. A recent report describes that integrin activity can be down-modulated by the c-Raf-1/MAP2 kinase pathway. Specific activation of the c-Raf-1/MAP2 kinases by cell-permeable peptides in skeletal muscle of rabbits causes degeneration of muscle fibers. Therefore, we conclude that in alpha(7) integrin-deficient mice, the continuous activation of c-Raf-1 kinase causes a permanent reduction of integrin activity diminishing integrin-dependent cell-matrix interactions and thereby contributing to the development of the dystrophic phenotype.
Li, Te-Mao; Fong, Yi-Chin; Liu, Shan-Chi; Chen, Po-Chun; Tang, Chih-Hsin
2013-01-01
Chondrosarcoma is a primary malignant bone cancer, with a potent capacity to invade locally and cause distant metastasis; it has a poor prognosis and shows a predilection for metastasis to the lungs. Brain derived neurotrophic factor (BDNF) is a small-molecule protein from the neurotrophin family of growth factors that is associated with the disease status and outcomes of cancers. However, the effect of BDNF on migration activity in human chondrosarcoma cells is mostly unknown. Here, we found that human chondrosarcoma tissues showed significant expression of BDNF, which was higher than that in normal cartilage and primary chondrocytes. We also found that BDNF increased the migration and expression of β5 integrin in human chondrosarcoma cells. In addition, knockdown of BDNF expression markedly inhibited migratory activity. BDNF-mediated migration and β5 integrin up-regulation were attenuated by antibody, inhibitor, or siRNA against the TrkB receptor. Pretreatment of chondrosarcoma cells with PI3K, Akt, and NF-κB inhibitors or mutants also abolished BDNF-promoted migration and integrin expression. The PI3K, Akt, and NF-κB signaling pathway was activated after BDNF treatment. Taken together, our results indicate that BDNF enhances the migration of chondrosarcoma by increasing β5 integrin expression through a signal transduction pathway that involves the TrkB receptor, PI3K, Akt, and NF-κB. BDNF thus represents a promising new target for treating chondrosarcoma metastasis. PMID:23874483
Flores-Robles, Donaciano; Rosales, Carlos; Rosales-Encina, José Luis; Talamás-Rohana, Patricia
2003-01-01
During tissue invasion, Entamoeba histolytica trophozoites interact with endothelial cells and extracellular matrix (ECM) proteins such as fibronectin (FN), collagen, and laminin. It has been demonstrated that trophozoites interact with FN through a beta1 integrin-like FN receptor (beta 1EhFNR), activating tyrosine kinases. In order to characterize the signaling process triggered by the amoebic receptor, activation, and association of tyrosine kinases and structural proteins were determined. As a result of FN binding by the beta 1EhFNR, the receptor itself, FAK, and paxillin were phosphorylated in tyrosine. Co-immunoprecipitation experiments showed that a multimolecular signaling complex was formed by the amoebic FN receptor, FAK, paxillin, and vinculin. These results strongly suggest that a signaling pathway, similar to the one used in mammalian cells, is activated when E. histolytica trophozoites adhere to FN.
Visualization of integrin Mac-1 in vivo.
Lim, Kihong; Hyun, Young-Min; Lambert-Emo, Kris; Topham, David J; Kim, Minsoo
2015-11-01
β2 integrins play critical roles in migration of immune cells and in the interaction with other cells, pathogens, and the extracellular matrix. Among the β2 integrins, Mac-1 (Macrophage antigen-1), composed of CD11b and CD18, is mainly expressed in innate immune cells and plays a major role in cell migration and trafficking. In order to image Mac-1-expressing cells both in live cells and mouse, we generated a knock-in (KI) mouse strain expressing CD11b conjugated with monomeric yellow fluorescent protein (mYFP). Expression of CD11b-mYFP protein was confirmed by Western blot and silver staining of CD11b-immunoprecipitates and total cell lysates from the mouse splenocytes. Mac-1-mediated functions of the KI neutrophils were comparable with those in WT cells. The fluorescence intensity of CD11b-mYFP was sufficient to image CD11b expressing cells in live mice using intravital two-photon microscopy. In vitro, dynamic changes in the intracellular localization of CD11b molecules could be measured by epifluorescent microscopy. Finally, CD11b-expressing immune cells from tissue were easily detected by flow cytometry without anti-CD11b antibody staining. Copyright © 2015 Elsevier B.V. All rights reserved.
Direct Thy-1/alphaVbeta3 integrin interaction mediates neuron to astrocyte communication.
Hermosilla, Tamara; Muñoz, Daniel; Herrera-Molina, Rodrigo; Valdivia, Alejandra; Muñoz, Nicolás; Nham, Sang-Uk; Schneider, Pascal; Burridge, Keith; Quest, Andrew F G; Leyton, Lisette
2008-06-01
Thy-1 is an abundant neuronal glycoprotein of poorly defined function. We recently provided evidence indicating that Thy-1 clusters a beta3-containing integrin in astrocytes to induce tyrosine phosphorylation, RhoA activation and the formation of focal adhesions and stress fibers. To date, the alpha subunit partner of beta3 integrin in DI TNC1 astrocytes is unknown. Similarly, the ability of neuronal, membrane-bound Thy-1 to trigger astrocyte signaling via integrin engagement remains speculation. Here, evidence that alphav forms an alphavbeta3 heterodimer in DI TNC1 astrocytes was obtained. In neuron-astrocyte association assays, the presence of either anti-alphav or anti-beta3 integrin antibodies reduced cell-cell interaction demonstrating the requirement of both integrin subunits for this association. Moreover, anti-Thy-1 antibodies blocked stimulation of astrocytes by neurons but not the binding of these two cell types. Thus, neuron-astrocyte association involved binding between molecular components in addition to the Thy-1-integrin; however, the signaling events leading to focal adhesion formation in astrocytes depended exclusively on the latter interaction. Additionally, wild-type (RLD) but not mutated (RLE) Thy-1 was shown to directly interact with alphavbeta3 integrin by Surface Plasmon Resonance analysis. This interaction was promoted by divalent cations and was species-independent. Together, these results demonstrate that the alphavbeta3 integrin heterodimer interacts directly with Thy-1 present on neuronal cells to stimulate astrocytes.
Wang, Ruifei; Bi, Jiajia; Ampah, Khamal Kwesi; Zhang, Chunmei; Li, Ziyi; Jiao, Yang; Wang, Xiaoru; Ba, Xueqing; Zeng, Xianlu
2013-08-01
Cell adhesion and spreading require integrins-mediated cell-extracellular matrix interaction. Integrins function through binding to extracellular matrix and subsequent clustering to initiate focal adhesion formation and actin cytoskeleton rearrangement. Lipid raft, a liquid ordered plasma membrane microdomain, has been reported to play major roles in membrane motility by regulating cell surface receptor function. Here, we identified that lipid raft integrity was required for β1 integrin-mediated initial spreading of melanoma A375 cells on fibronectin. We found that lipid raft disruption with methyl-β-cyclodextrin led to the inability of focal adhesion formation and actin cytoskeleton rearrangement by preventing β1 integrin clustering. Furthermore, we explored the possible mechanism by which lipid raft regulates β1 integrin clustering and demonstrated that intact lipid raft could recruit and modify some adaptor proteins, such as talin, α-actinin, vinculin, paxillin and FAK. Lipid raft could regulate the location of these proteins in lipid raft fractions and facilitate their binding to β1 integrin, which may be crucial for β1 integrin clustering. We also showed that lipid raft disruption impaired A375 cell migration in both transwell and wound healing models. Together, these findings provide a new insight for the relationship between lipid raft and the regulation of integrins. Copyright © 2013 Elsevier Ltd. All rights reserved.
Keszthelyi, E; Karlik, S; Hyduk, S; Rice, G P; Gordon, G; Yednock, T; Horner, H
1996-10-01
The leukocyte integrin receptor, alpha 4 beta 1, and its endothelial cell ligand, vascular cell adhesion molecule 1, appear to be of critical importance in the leukocyte trafficking that accompanies CNS damage in experimental allergic encephalomyelitis (EAE). In this study, the persistence of the role for alpha 4 beta 1/VCAM-1 in EAE was established by observing antibody-mediated disease reversal up to 1 month following disease onset. Limited treatment with a monoclonal antibody against alpha 4 integrin, GG5/3, resulted in a significant decrease in both clinical and histopathologic signs. This was not observed in isotype control experiments. In the latter phase of progressive disease, widespread demyelination occurred in the animals that did not respond to 6 days of anti-alpha 4 treatment. These results demonstrate an essential role for alpha 4 beta 1 interactions throughout active EAE and illustrate the difference between reversible clinical deficits caused by edema and irreversible deficits associated with demyelination.
Freud-2/CC2D1B mediates dual repression of the serotonin-1A receptor gene.
Hadjighassem, Mahmoud R; Galaraga, Kimberly; Albert, Paul R
2011-01-01
The serotonin-1A (5-HT1A) receptor functions as a pre-synaptic autoreceptor in serotonin neurons that regulates their activity, and is also widely expressed on non-serotonergic neurons as a post-synaptic heteroreceptor to mediate serotonin action. The 5-HT1A receptor gene is strongly repressed by a dual repressor element (DRE), which is recognized by two proteins: Freud-1/CC2D1A and another unknown protein. Here we identify mouse Freud-2/CC2D1B as the second repressor of the 5-HT1A-DRE. Freud-2 shares 50% amino acid identity with Freud-1, and contains conserved structural domains. Mouse Freud-2 bound specifically to the rat 5-HT1A-DRE adjacent to, and partially overlapping, the Freud-1 binding site. By supershift assay using nuclear extracts from L6 myoblasts, Freud-2-DRE complexes were distinguished from Freud-1-DRE complexes. Freud-2 mRNA and protein were detected throughout mouse brain and peripheral tissues. Freud-2 repressed 5-HT1A promoter-reporter constructs in a DRE-dependent manner in non-neuronal (L6) or 5-HT1A-expressing neuronal (NG108-15, RN46A) cell models. In NG108-15 cells, knockdown of Freud-2 using a specific short-interfering RNA reduced endogenous Freud-2 protein levels and decreased Freud-2 bound to the 5-HT1A-DRE as detected by chromatin immunoprecipitation assay, but increased 5-HT1A promoter activity and 5-HT1A protein levels. Taken together, these data show that Freud-2 is the second component that, with Freud-1, mediates dual repression of the 5-HT1A receptor gene at the DRE. © 2010 The Authors. European Journal of Neuroscience © 2010 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.
LENUS (Irish Health Repository)
Wakai, A
2012-02-03
The effects of adenosine on neutrophil (polymorphonuclear neutrophils; PMN)-directed changes in vascular permeability are poorly characterized. This study investigated whether adenosine modulates activated PMN vascular endothelial growth factor (vascular permeability factor; VEGF) release and transendothelial migration. PMN activated with tumour necrosis factor-alpha (TNF-alpha, 10 ng\\/mL) were incubated with adenosine and its receptor-specific analogues. Culture supernatants were assayed for VEGF. PMN transendothelial migration across human umbilical vein endothelial cell (HUVEC) monolayers was assessed in vitro. Adhesion molecule receptor expression was assessed flow cytometrically. Adenosine and some of its receptor-specific analogues dose-dependently inhibited activated PMN VEGF release. The rank order of potency was consistent with the affinity profile of human A2B receptors. The inhibitory effect of adenosine was reversed by 3,7-dimethyl-1-propargylxanthine, an A2 receptor antagonist. Adenosine (100 microM) or the A2B receptor agonist 5\\'-N-ethylcarboxamidoadenosine (NECA, 100 microM) significantly reduced PMN transendothelial migration. However, expression of activated PMN beta2 integrins and HUVEC ICAM-1 were not significantly altered by adenosine or NECA. Adenosine attenuates human PMN VEGF release and transendothelial migration via the A2B receptor. This provides a novel target for the modulation of PMN-directed vascular hyperpermeability in conditions such as the capillary leak syndrome.
Guha, Prajna; Cunetta, Marissa; Somasundar, Ponnandai; Espat, N Joseph; Junghans, Richard P; Katz, Steven C
2017-08-01
Chimeric antigen receptor expressing T cells (CAR-T) are a promising form of immunotherapy, but the influence of age-related immune changes on CAR-T production remains poorly understood. We showed that CAR-T cells from geriatric donors (gCAR-T) are functionally impaired relative to CAR-T from younger donors (yCAR-T). Higher transduction efficiencies and improved cell expansion were observed in yCAR-T cells compared with gCAR-T. yCAR-T demonstrated significantly increased levels of proliferation and signaling activation of phosphorylated (p)Erk, pAkt, pStat3, and pStat5. Furthermore, yCAR-T contained higher proportions of CD4 and CD8 effector memory (EM) cells, which are known to have enhanced cytolytic capabilities. Accordingly, yCAR-T demonstrated higher levels of tumor antigen-specific cytotoxicity compared with gCAR-T. Enhanced tumor killing by yCAR-T correlated with increased levels of perforin and granzyme B. yCAR-T had increased α5β1 integrin expression, a known mediator of retroviral transduction. We found that treatment with M-CSF or TGF-β1 rescued the impaired transduction efficiency of the gCAR-T by increasing the α5β1 integrin expression. Neutralization of α5β1 confirmed that this integrin was indispensable for CAR expression. Our study suggests that the increase of α5β1 integrin expression levels enhances CAR expression and thereby improves tumor killing by gCAR-T. © Society for Leukocyte Biology.
LENUS (Irish Health Repository)
Andrews, E J
2012-02-03
BACKGROUND: Recent studies have demonstrated that metastatic disease develops from tumor cells that adhere to endothelial cells and proliferate intravascularly. The beta-1 integrin family and its ligand laminin have been shown to be important in tumor-to-endothelial cell adhesion. Lipopolysaccharide (LPS) has been implicated in the increased metastatic tumor growth that is seen postoperatively. We postulated that LPS increases tumor cell expression of beta-1 integrins and that this leads to increased adhesion. METHODS: The human metastatic colon cancer cell line LS174T was labeled with an enhanced green fluorescent protein (eGFP) using retroviral transfection. Cell cultures were treated with LPS for 1, 2, and 4 h (n = 6 each) and were subsequently cocultured for 30 or 120 min with confluent human umbilical vein endothelial cells (HUVECs), to allow adherence. Adherent tumor cells were counted using fluorescence microscopy. These experiments were carried out in the presence or absence of a functional blocking beta-1 integrin monoclonal antibody (4B4). Expression of beta-1 integrin and laminin on tumor and HUVECs was assessed using flow cytometric analysis. Tumor cell NF-kappaB activation after incubation with LPS was measured. RESULTS: Tumor cell and HUVEC beta-1 integrin expression and HUVEC expression of laminin were significantly (P < 0.05) enhanced after incubation with LPS. Tumor cell adhesion to HUVECs was significantly increased. Addition of the beta-1 integrin blocking antibody reduced tumor cell adhesion to control levels. LPS increased tumor cell NF-kappaB activation. CONCLUSIONS: Exposure to LPS increases tumor cell adhesion to the endothelium through a beta-1 integrin-mediated pathway that is NF-kappaB dependent. This may provide a target for immunotherapy directed at reducing postoperative metastatic tumor growth.
Modulation of type II TGF-β receptor degradation by integrin-linked kinase.
Vi, Linda; Boo, Stellar; Sayedyahossein, Samar; Singh, Randeep K; McLean, Sarah; Di Guglielmo, Gianni M; Dagnino, Lina
2015-03-01
Cutaneous responses to injury, infection, and tumor formation involve the activation of resident dermal fibroblasts and subsequent transition to myofibroblasts. The key for induction of myofibroblast differentiation is the activation of transforming growth factor-β (TGF-β) receptors and stimulation of integrins and their associated proteins, including integrin-linked kinase (ILK). Cross-talk processes between TGF-β and ILK are crucial for myofibroblast formation, as ILK-deficient dermal fibroblasts exhibit impaired responses to TGF-β receptor stimulation. We now show that ILK associates with type II TGF-β receptors (TβRII) in ligand- and receptor kinase activity-independent manners. In cells with targeted Ilk gene inactivation, cellular levels of TβRII are decreased, through mechanisms that involve enhanced ubiquitination and proteasomal degradation. Partitioning of TGF-β receptors into membrane has been linked to proteasome-dependent receptor degradation. We found that interfering with membrane raft formation in ILK-deficient cells restored TβRII levels and signaling. These observations support a model whereby ILK functions in fibroblasts to direct TβRII away from degradative pathways during their differentiation into myofibroblasts.
Energy Technology Data Exchange (ETDEWEB)
Carpenter, R D; Natarajan, A; Lau, E Y; Andrei, M; Solano, D M; Lightstone, F C; DeNardo, S J; Lam, K S; Kurth, M J
2010-02-08
The cell surface receptor {alpha}{sub 4}{beta}{sub 1} integrin is an attractive yet poorly understood target for selective diagnosis and treatment of T- and B-cell lymphomas. This report focuses on the rapid microwave preparation of medicinally pertinent benzimidazole heterocycles, structure-activity relationships (SAR) of novel halobenzimidazole carboxamide antagonists 3-6, and preliminary biological evaluation of radioiodinated agents 7, 8, and 18. The I-125 derivative 18 had good tumor uptake (12 {+-} 1% ID/g at 24 h; 4.5 {+-} 1% ID/g at 48 h) and tumor:kidney ratio ({approx}4:1 at 24 h; 2.5:1 at 48 h) in xenograft murine models of B-cell lymphoma. Molecular homology models of {alpha}{sub 4}{beta}{sub 1} integrin have predicted that docked halobenzimidazole carboxamides have the halogen atom in a suitable orientation for halogen-hydrogen bonding. These high affinity ({approx} pM binding) halogenated ligands are attractive tools for medicinal and biological use; the fluoro and iodo derivatives are potential radiodiagnostic ({sup 18}F) or radiotherapeutic ({sup 131}I) agents, whereas the chloro and bromo analogues could provide structural insight into integrin-ligand interactions through photoaffinity cross-linking/mass spectroscopy experiments, as well as co-crystallization X-ray studies.
Directory of Open Access Journals (Sweden)
Hai-Yan Han
Full Text Available αν and β1 integrins mediate Aβ-induced neurotoxicity in primary hippocampal neurons. We treated hippocampal neurons with 2.5 µg/mL 17E6 and 5 µg/mL ab58524, which are specific αν and β1 integrin antagonists, respectively, for 42 h prior to 10 µM Aβ treatment. Next, we employed small interfering RNA (siRNA to silence focal adhesion kinase (FAK, a downstream target gene of integrins. The siRNAs were designed with a target sequence, an MOI of 10 and the addition of 5 µg/mL polybrene. Under these conditions, the neurons were transfected and the apoptosis of different cell types was detected. Moreover, we used real-time PCR and Western blotting analyses to detect the expression of FAK and ρFAK genes in different cell types and investigated the underlying mechanism and signal pathway by which αν and β1 integrins mediate Aβ-induced neurotoxicity in hippocampal neurons. An MTT assay showed that both 17E6 and ab58524 significantly increased cell viability compared with the Aβ-treated neurons (P<0.01 and P<0.05, respectively. However, this protective effect was markedly attenuated after transfection with silencing FAK (siFAK. Moreover, TUNEL immunostaining and flow cytometry indicated that both 17E6 and ab58524 significantly protected hippocampal neurons against apoptosis induced by Aβ (P<0.05 compared with the Aβ-treated cells. However, this protective effect was reversed with siFAK treatment. Both the gene and protein expression of FAK increased after Aβ treatment. Interestingly, as the gene and protein levels of FAK decreased, the ρFAK protein expression markedly increased. Furthermore, both the gene and protein expression of FAK and ρFAK were significantly diminished. Thus, we concluded that both αν and β1 integrins interfered with Aβ-induced neurotoxicity in hippocampal neurons and that this mechanism partially contributes to the activation of the Integrin-FAK signaling pathway.
Laminin-binding integrins and their tetraspanin partners as potential antimetastatic targets
Stipp, Christopher S.
2010-01-01
Within the integrin family of cell adhesion receptors, integrins α3β1, α6β1, α6β4 and α7β1 make up a laminin-binding subfamily. The literature is divided on the role of these laminin-binding integrins in metastasis, with different studies indicating either pro- or antimetastatic functions. The opposing roles of the laminin-binding integrins in different settings might derive in part from their unusually robust associations with tetraspanin proteins. Tetraspanins organise integrins into multiprotein complexes within discrete plasma membrane domains termed tetraspanin-enriched microdomains (TEMs). TEM association is crucial to the strikingly rapid cell migration mediated by some of the laminin-binding integrins. However, emerging data suggest that laminin-binding integrins also promote the stability of E-cadherin-based cell–cell junctions, and that tetraspanins are essential for this function as well. Thus, TEM association endows the laminin-binding integrins with both pro-invasive functions (rapid migration) and anti-invasive functions (stable cell junctions), and the composition of TEMs in different cell types might help determine the balance between these opposing activities. Unravelling the tetraspanin control mechanisms that regulate laminin-binding integrins will help to define the settings where inhibiting the function of these integrins would be helpful rather than harmful, and may create opportunities to modulate integrin activity in more sophisticated ways than simple functional blockade. PMID:20078909
Lonsdorf, Anke S.; Krämer, Björn F.; Fahrleitner, Manuela; Schönberger, Tanja; Gnerlich, Stephan; Ring, Sabine; Gehring, Sarah; Schneider, Stefan W.; Kruhlak, Michael J.; Meuth, Sven G.; Nieswandt, Bernhard; Gawaz, Meinrad; Enk, Alexander H.; Langer, Harald F.
2012-01-01
A mutual relationship exists between metastasizing tumor cells and components of the coagulation cascade. The exact mechanisms as to how platelets influence blood-borne metastasis, however, remain poorly understood. Here, we used murine B16 melanoma cells to observe functional aspects of how platelets contribute to the process of hematogenous metastasis. We found that platelets interfere with a distinct step of the metastasis cascade, as they promote adhesion of melanoma cells to the endothelium in vitro under shear conditions. Constitutively active platelet receptor GPIIb/IIIa (integrin αIIbβ3) expressed on Chinese hamster ovary cells promoted melanoma cell adhesion in the presence of fibrinogen, whereas blocking antibodies to aνβ3 integrin on melanoma cells or to GPIIb/IIIa significantly reduced melanoma cell adhesion to platelets. Furthermore, using intravital microscopy, we observed functional platelet-melanoma cell interactions, as platelet depletion resulted in significantly reduced melanoma cell adhesion to the injured vascular wall in vivo. Using a mouse model of hematogenous metastasis to the lung, we observed decreased metastasis of B16 melanoma cells to the lung by treatment with a mAb blocking the aν subunit of aνβ3 integrin. This effect was significantly reduced when platelets were depleted in vivo. Thus, the engagement of GPIIb/IIIa with aνβ3 integrin interaction mediates tumor cell-platelet interactions and highlights how this interaction is involved in hematogenous tumor metastasis. PMID:22102277
Highly Potent, Water Soluble Benzimidazole Antagonist for Activated (alpha)4(beta)1 Integrin
Energy Technology Data Exchange (ETDEWEB)
Carpenter, R D; Andrei, M; Lau, E Y; Lightstone, F C; Liu, R; Lam, K S; Kurth, M J
2007-08-29
The cell surface receptor {alpha}{sub 4}{beta}{sub 1} integrin, activated constitutively in lymphoma, can be targeted with the bisaryl urea peptidomimetic antagonist 1 (LLP2A). However, concerns on its preliminary pharmacokinetic (PK) profile provided an impetus to change the pharmacophore from a bisaryl urea to a 2-arylaminobenzimidazole moiety resulting in improved solubility while maintaining picomolar potency [5 (KLCA4); IC{sub 50} = 305 pM]. With exceptional solubility, this finding has potential for improving PK to help diagnose and treat lymphomas.
Activation of the kinin B1 receptor attenuates melanoma tumor growth and metastasis.
Directory of Open Access Journals (Sweden)
Patricia Dillenburg-Pilla
Full Text Available Melanoma is a very aggressive tumor that does not respond well to standard therapeutic approaches, such as radio- and chemotherapies. Furthermore, acquiring the ability to metastasize in melanoma and many other tumor types is directly related to incurable disease. The B1 kinin receptor participates in a variety of cancer-related pathophysiological events, such as inflammation and angiogenesis. Therefore, we investigated whether this G protein-coupled receptor plays a role in tumor progression. We used a murine melanoma cell line that expresses the kinin B1 receptor and does not express the kinin B2 receptor to investigate the precise contribution of activation of the B1 receptor in tumor progression and correlated events using various in vitro and in vivo approaches. Activation of the kinin B1 receptor in the absence of B2 receptor inhibits cell migration in vitro and decreases tumor formation in vivo. Moreover, tumors formed from cells stimulated with B1-specific agonist showed several features of decreased aggressiveness, such as smaller size and infiltration of inflammatory cells within the tumor area, higher levels of pro-inflammatory cytokines implicated in the host anti-tumor immune response, lower number of cells undergoing mitosis, a poorer vascular network, no signs of invasion of surrounding tissues or metastasis and increased animal survival. Our findings reveal that activation of the kinin B1 receptor has a host protective role during murine melanoma tumor progression, suggesting that the B1 receptor could be a new anti-tumor GPCR and provide new opportunities for therapeutic targeting.
Integrin-linked kinase is required for TGF-β1 induction of dermal myofibroblast differentiation.
Vi, Linda; de Lasa, Cristina; DiGuglielmo, Gianni M; Dagnino, Lina
2011-03-01
Cutaneous repair after injury requires activation of resident dermal fibroblasts and their transition to myofibroblasts. The key stimuli for myofibroblast formation are activation of transforming growth factor-β (TGF-β) receptors and mechanotransduction mediated by integrins and associated proteins. We investigated the role of integrin-linked kinase (ILK) in TGF-β1 induction of dermal fibroblast transition to myofibroblasts. ILK-deficient fibroblasts treated with TGF-β1 exhibited attenuation of Smad 2 and 3 phosphorylation, accompanied by impaired transcriptional activation of Smad targets, such as α-smooth muscle actin. These alterations were not limited to Smad-associated TGF-β1 responses, as stimulation of noncanonical mitogen-activated protein kinase pathways by this growth factor was also diminished in the absence of ILK. ILK-deficient fibroblasts exhibited abnormalities in the actin cytoskeleton, and did not form supermature focal adhesions or contractile F-actin stress fibers, indicating a severe impairment in their capacity to differentiate into myofibroblasts. These defects extended to the inability of cells to contract extracellular matrices when embedded in collagen lattices. We conclude that ILK is necessary to transduce signals implicated in the transition of dermal fibroblasts to myofibroblasts originating from matrix substrates and TGF-β1.
Vehlow, Anne; Klapproth, Erik; Storch, Katja; Dickreuter, Ellen; Seifert, Michael; Dietrich, Antje; Bütof, Rebecca; Temme, Achim; Cordes, Nils
2017-07-25
Resistance of cancer stem-like and cancer tumor bulk cells to radiochemotherapy and destructive infiltration of the brain fundamentally influence the treatment efficiency to cure of patients suffering from Glioblastoma (GBM). The interplay of adhesion and stress-related signaling and activation of bypass cascades that counteract therapeutic approaches remain to be identified in GBM cells. We here show that combined inhibition of the adhesion receptor β1 integrin and the stress-mediator c-Jun N-terminal kinase (JNK) induces radiosensitization and blocks invasion in stem-like and patient-derived GBM cultures as well as in GBM cell lines. In vivo, this treatment approach not only significantly delays tumor growth but also increases median survival of orthotopic, radiochemotherapy-treated GBM mice. Both, in vitro and in vivo, effects seen with β1 integrin/JNK co-inhibition are superior to the monotherapy. Mechanistically, the in vitro radiosensitization provoked by β1 integrin/JNK targeting is caused by defective DNA repair associated with chromatin changes, enhanced ATM phosphorylation and prolonged G2/M cell cycle arrest. Our findings identify a β1 integrin/JNK co-dependent bypass signaling for GBM therapy resistance, which might be therapeutically exploitable.
Distinct roles for dystroglycan, beta1 integrin and perlecan in cell surface laminin organization
DEFF Research Database (Denmark)
Henry, M D; Satz, J S; Brakebusch, C
2001-01-01
Dystroglycan (DG) is a cell surface receptor for several extracellular matrix (ECM) molecules including laminins, agrin and perlecan. Recent data indicate that DG function is required for the formation of basement membranes in early development and the organization of laminin on the cell surface...... integrin-deficient ES cells, laminin-1 binds to the cell surface, but fails to organize into more morphologically complex structures. This result indicates that beta1 integrin function is required after DG function in the cell surface-mediated laminin assembly process. In perlecan-deficient ES cells......, the formation of complex laminin-1 structures is defective, implicating perlecan in the laminin matrix assembly process. Moreover, laminin and perlecan reciprocally modulate the organization of the other on the cell surface. Taken together, the data support a model whereby DG serves as a receptor essential...
Energy Technology Data Exchange (ETDEWEB)
Kovalova, Natalia, E-mail: kovalova@msu.edu [Department of Pharmacology and Toxicology, Michigan State University, Lansing, MI 48824 (United States); Institute for Integrative Toxicology, Michigan State University, Lansing, MI 48824 (United States); Manzan, Maria, E-mail: ale.manzan@gmail.com [Institute for Integrative Toxicology, Michigan State University, Lansing, MI 48824 (United States); Crawford, Robert, E-mail: crawfo28@msu.edu [Institute for Integrative Toxicology, Michigan State University, Lansing, MI 48824 (United States); Kaminski, Norbert, E-mail: kamins11@msu.edu [Department of Pharmacology and Toxicology, Michigan State University, Lansing, MI 48824 (United States); Institute for Integrative Toxicology, Michigan State University, Lansing, MI 48824 (United States)
2016-10-15
Previous studies have demonstrated that most of the intraspecies variation in sensitivity to the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), including suppression of antibody responses, in murine models is due to single nucleotide polymorphisms (SNPs) within the aryl hydrocarbon receptor (AhR) gene. The underlying reason for variation in sensitivity to TCDD-induced suppression of IgM responses among humans is not well understood, but is thought, in part, to be a result of different polymorphic forms of the AhR expressed by different individuals. In this study, the functional properties of six (P517S, R554K, V570I, V570I + P517S, R554K + V570I and P517S + R554K + V570I) human AhR variants were examined in the human B cell line, SKW 6.4. TCDD-induced Cyp1B1 and Cyp1A2 mRNA expression levels and Cyp1B1-regulated reporter gene activity, used for comparative purposes, were markedly lower in SKW cells containing the R554K SNP than in SKW-AHR{sup +} (control AhR) cells. Furthermore, all AhR variants were able to mediate TCDD-induced suppression of the IgM response; however, a combined P517S + R554K + V570I variant partially reduced sensitivity to TCDD-mediated suppression of IgM secretion. Collectively, our findings show that the R554K human AhR SNP alone altered sensitivity of human B cells to TCDD-mediated induction of Cyp1B1 and Cyp1A2. By contrast, attenuation of TCDD-induced IgM suppression required a combination of all three SNPs P517S, R554K, and V570I. - Highlights: • Mouse, rat and SKW-AHR{sup +} B cells have a similar window of sensitivity to TCDD. • R554K AhR SNP alters B cell sensitivity to TCDD-mediated Cyp1B1 and Cyp1A2 induction. • Combination of P517S, R554K, and V570I SNPs attenuates TCDD-induced IgM suppression.
DEFF Research Database (Denmark)
Xu, Ruodan; Povlsen, Gro Klitgaard; Soroka, Vladislav
2010-01-01
The epidermal growth factor family of receptor tyrosine kinases (ErbBs) plays essential roles in tumorigenesis and cancer disease progression, and therefore has become an attractive target for structure-based drug design. ErbB receptors are activated by ligand-induced homo- and heterodimerization...... constitutes part of the dimerization arm of ErbB3. Inherbin3 binds to the extracellular domains of all four ErbB receptors, with the lowest peptide binding affinity for ErbB4. Inherbin3 functions as an antagonist of epidermal growth factor (EGF)-ErbB1 signaling. We show that Inherbin3 inhibits EGF-induced Erb....... Structural studies have revealed that ErbB receptor dimers are stabilized by receptor-receptor interactions, primarily mediated by a region in the second extracellular domain, termed the "dimerization arm". The present study is the first biological characterization of a peptide, termed Inherbin3, which...
CXCL1 and CXCL2 Regulate NLRP3 Inflammasome Activation via G-Protein-Coupled Receptor CXCR2.
Boro, Monoranjan; Balaji, Kithiganahalli Narayanaswamy
2017-09-01
Inflammation is an extensively concerted process that confers protection to the host encountering immune insult. The major inflammatory mediators include IL-1 family members, such as IL-1β, and the functional activation of such molecules is arbitrated by their regulated cleavage brought about by components of a multiprotein complex called inflammasome. In this context, NLR family pyrin domain containing 3 (NLRP3) inflammasome activation often acts as a rate-limiting step in regulating critical cell-fate decisions in various inflammatory scenarios. In this study, we identify the G-protein-coupled receptor CXCR2 (recognizing chemokines CXCL1 and CXCL2) as another arm feeding into the regulated activation of NLRP3 inflammasome in macrophages. We demonstrate that in vivo blocking of CXCL1 and CXCL2 can significantly reduce the Mycobacterium tuberculosis -induced bioactive IL-1β production. Further, CXCL1 could amplify the inflammasome activation in in vivo mouse models of carrageenan-induced inflammation in footpads and air pouches. The mechanistic insights revealed CXCR2-driven protein kinase C μ-dependent integrin-linked kinase to be essential for CXCL1-mediated activation of NLRP3 inflammasome. Blocking the activity of integrin-linked kinase or protein kinase C μ either by small interfering RNA-mediated knockdown or pharmacological inhibitor compromised inflammasome activation and subsequent production of bioactive IL-1β. Taken together, our study demonstrates CXCR2-driven activation of NLRP3 inflammasome in macrophages and indicates a potential host-directed therapeutic target to limit the damaging inflammation associated with overt production of proinflammatory IL-1β. Copyright © 2017 by The American Association of Immunologists, Inc.
Kelley, Melissa D; Phomakay, Raynin; Lee, Madison; Niedzwiedz, Victoria; Mayo, Rachel
2017-01-01
The interplay between cellular adhesion and proliferation is complex; however, integrins, particularly the α5β1 subset, play a pivotal role in orchestrating critical cellular signals that culminate in cellular adhesion and growth. Retinoids modify the expression of a variety of adhesive/proliferative signaling proteins including α5β1 integrins; however, the role of specific retinoic acid receptors involved in these processes has not been elucidated. In this study, the effect of all-trans-retinoic acid receptor (RAR) agonists on K562 cellular adhesion, proliferation, and α5β1 integrin cell surface expression was investigated. RARγ agonist exposure increased K562 cellular adhesion to RGD containing extracellular matrix proteins fibronectin and FN-120 in a time- and concentration dependent manner, while RARα or RARβ agonist treatment had no effect on cellular adhesion. Due to the novel RARγ- dependent cellular adhesion response exhibited by K562 cells, we examined α5 and β1 integrin subunit expression when K562 cells were exposed to retinoid agonists or vehicle for 24, 48, 72 or 96 hours. Our data demonstrates no differences in K562 cell surface expression of the α5 integrin subunit when cells were exposed to RARα, RARβ, or RARγ agonists for all time points tested. In contrast, RARγ agonist exposure resulted in an increase in cell surface β1 integrin subunit expression within 48 hours that was sustained at 72 and 96 hours. Finally, we demonstrate that while exposure to RARα or RARβ agonists have no effect on K562 cellular proliferation, the RARγ agonist significantly dampens K562 cellular proliferation levels in a time- and concentration- dependent manner. Our study is the first to report that treatment with a RARγ specific agonist augments cellular adhesion to α5β1 integrin substrates, increases cell surface levels of the β1 integrin subunit, and dampens cellular proliferation in a time and concentration dependent manner in a human
Collagen Type I as a Ligand for Receptor-Mediated Signaling
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Iris Boraschi-Diaz
2017-05-01
Full Text Available Collagens form the fibrous component of the extracellular matrix in all multi-cellular animals. Collagen type I is the most abundant collagen present in skin, tendons, vasculature, as well as the organic portion of the calcified tissue of bone and teeth. This review focuses on numerous receptors for which collagen acts as a ligand, including integrins, discoidin domain receptors DDR1 and 2, OSCAR, GPVI, G6b-B, and LAIR-1 of the leukocyte receptor complex (LRC and mannose family receptor uPARAP/Endo180. We explore the process of collagen production and self-assembly, as well as its degradation by collagenases and gelatinases in order to predict potential temporal and spatial sites of action of different collagen receptors. While the interactions of the mature collagen matrix with integrins and DDR are well-appreciated, potential signals from immature matrix as well as collagen degradation products are possible but not yet described. The role of multiple collagen receptors in physiological processes and their contribution to pathophysiology of diseases affecting collagen homeostasis require further studies.
Newman-Tancredi, Adrian; Cussac, Didier; Marini, Laetitia; Touzard, Manuelle; Millan, Mark J
2003-03-01
1. Serotonin 5-HT(1B) receptors couple to G-proteins of the Gi/o family. However, their activation of specific G-protein subtypes is poorly characterised. Using an innovative antibody capture/guanosine-5'-0-(3-[(35)S]thio)-triphosphate ([(35)S]GTPgammaS) binding strategy, we characterised Galpha(i3) subunit activation by h5-HT(1B) receptors stably expressed in Chinese hamster ovary (CHO) cells. 2. The agonists, 5-HT, alniditan and BMS181,101, stimulated Galpha(i3), whereas methiothepin and SB224,289 behaved as inverse agonists. The selective 5-HT(1B) receptor ligand, S18127, modestly stimulated Galpha(i3) and reversed the actions of both 5-HT and methiothepin. S18127 (1 micro M) also produced parallel, dextral shifts of the 5-HT and methiothepin isotherms. 3. Isotopic dilution experiments ([(35)S]GTPgammaS versus GTPgammaS) revealed high-affinity [(35)S]GTPgammaS binding to Galpha(i3) subunits in the absence of receptor ligands indicating constitutive activity. High-affinity [(35)S]GTPgammaS binding was increased 2.8-fold by 5-HT with an increase in the affinity of GTPgammaS for Galpha(i3) subunits. In contrast, methiothepin halved the number of high-affinity binding sites and decreased their affinity. 4. h5-HT(1B) receptor-mediated Galpha(i3) subunit activation was dependent on the concentration of NaCl. At 300 mM, 5-HT stimulated [(35)S]GTPgammaS binding, basal Galpha(i3) activation was low and methiothepin was inactive. In contrast, at 10 mM NaCl, basal activity was enhanced and the inverse agonist activity of methiothepin was accentuated. Under these conditions, 5-HT decreased Galpha(i3) activation. 5. In conclusion, at h5-HT(1B) receptors expressed in CHO cells: (i) inverse agonist induced inhibition of Galpha(i3), and its reversal by S18127, reveals constitutive activation of this Galpha subunit; (ii) constitutive Galpha(i3) activation can be quantified by isotopic dilution [(35)S]GTPgammaS binding and (iii) decreasing NaCl concentrations enhances Galpha(i3
Integrins as Therapeutic Targets: Successes and Cancers
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Sabine Raab-Westphal
2017-08-01
Full Text Available Integrins are transmembrane receptors that are central to the biology of many human pathologies. Classically mediating cell-extracellular matrix and cell-cell interaction, and with an emerging role as local activators of TGFβ, they influence cancer, fibrosis, thrombosis and inflammation. Their ligand binding and some regulatory sites are extracellular and sensitive to pharmacological intervention, as proven by the clinical success of seven drugs targeting them. The six drugs on the market in 2016 generated revenues of some US$3.5 billion, mainly from inhibitors of α4-series integrins. In this review we examine the current developments in integrin therapeutics, especially in cancer, and comment on the health economic implications of these developments.
Integrin-directed modulation of macrophage responses to biomaterials.
Zaveri, Toral D; Lewis, Jamal S; Dolgova, Natalia V; Clare-Salzler, Michael J; Keselowsky, Benjamin G
2014-04-01
Macrophages are the primary mediator of chronic inflammatory responses to implanted biomaterials, in cases when the material is either in particulate or bulk form. Chronic inflammation limits the performance and functional life of numerous implanted medical devices, and modulating macrophage interactions with biomaterials to mitigate this response would be beneficial. The integrin family of cell surface receptors mediates cell adhesion through binding to adhesive proteins nonspecifically adsorbed onto biomaterial surfaces. In this work, the roles of integrin Mac-1 (αMβ2) and RGD-binding integrins were investigated using model systems for both particulate and bulk biomaterials. Specifically, the macrophage functions of phagocytosis and inflammatory cytokine secretion in response to a model particulate material, polystyrene microparticles were investigated. Opsonizing proteins modulated microparticle uptake, and integrin Mac-1 and RGD-binding integrins were found to control microparticle uptake in an opsonin-dependent manner. The presence of adsorbed endotoxin did not affect microparticle uptake levels, but was required for the production of inflammatory cytokines in response to microparticles. Furthermore, it was demonstrated that integrin Mac-1 and RGD-binding integrins influence the in vivo foreign body response to a bulk biomaterial, subcutaneously implanted polyethylene terephthalate. A thinner foreign body capsule was formed when integrin Mac-1 was absent (~30% thinner) or when RGD-binding integrins were blocked by controlled release of a blocking peptide (~45% thinner). These findings indicate integrin Mac-1 and RGD-binding integrins are involved and may serve as therapeutic targets to mitigate macrophage inflammatory responses to both particulate and bulk biomaterials. Copyright © 2014 Elsevier Ltd. All rights reserved.
van Spriel, A B; Leusen, J H; van Egmond, M; Dijkman, H B; Assmann, K J; Mayadas, T N; van de Winkel, J G
2001-04-15
Receptors for human immunoglobulin (Ig)G and IgA initiate potent cytolysis of antibody (Ab)-coated targets by polymorphonuclear leukocytes (PMNs). Mac-1 (complement receptor type 3, CD11b/CD18) has previously been implicated in receptor cooperation with Fc receptors (FcRs). The role of Mac-1 in FcR-mediated lysis of tumor cells was characterized by studying normal human PMNs, Mac-1-deficient mouse PMNs, and mouse PMNs transgenic for human FcR. All PMNs efficiently phagocytosed Ab-coated particles. However, antibody-dependent cellular cytotoxicity (ADCC) was abrogated in Mac-1(-/-) PMNs and in human PMNs blocked with anti-Mac-1 monoclonal Ab (mAb). Mac-1(-/-) PMNs were unable to spread on Ab-opsonized target cells and other Ab-coated surfaces. Confocal laser scanning and electron microscopy revealed a striking difference in immunologic synapse formation between Mac-1(-/-) and wild-type PMNs. Also, respiratory burst activity could be measured outside membrane-enclosed compartments by using Mac-1(-/-) PMNs bound to Ab-coated tumor cells, in contrast to wild-type PMNs. In summary, these data document an absolute requirement of Mac-1 for FcR-mediated PMN cytotoxicity toward tumor targets. Mac-1(-/-) PMNs exhibit defective spreading on Ab-coated targets, impaired formation of immunologic synapses, and absent tumor cytolysis.
Lee, Hye Shin; Cheerathodi, Mujeeburahiman; Chaki, Sankar P.; Reyes, Steve B.; Zheng, Yanhua; Lu, Zhimin; Paidassi, Helena; DerMardirossian, Celine; Lacy-Hulbert, Adam; Rivera, Gonzalo M.
2015-01-01
Directional cell motility is essential for normal development and physiology, although how motile cells spatiotemporally activate signaling events remains largely unknown. Here, we have characterized an adhesion and signaling unit comprised of protein tyrosine phosphatase (PTP)-PEST and the extracellular matrix (ECM) adhesion receptor β8 integrin that plays essential roles in directional cell motility. β8 integrin and PTP-PEST form protein complexes at the leading edge of migrating cells and balance patterns of Rac1 and Cdc42 signaling by controlling the subcellular localization and phosphorylation status of Rho GDP dissociation inhibitor 1 (RhoGDI1). Translocation of Src-phosphorylated RhoGDI1 to the cell's leading edge promotes local activation of Rac1 and Cdc42, whereas dephosphorylation of RhoGDI1 by integrin-bound PTP-PEST promotes RhoGDI1 release from the membrane and sequestration of inactive Rac1/Cdc42 in the cytoplasm. Collectively, these data reveal a finely tuned regulatory mechanism for controlling signaling events at the leading edge of directionally migrating cells. PMID:25666508
Phospho-Tyrosine(s) vs. Phosphatidylinositol Binding in Shc Mediated Integrin Signaling
Lin, Xiaochen; Vinogradova, Olga
2015-01-01
The Shc adaptor protein, particularly its p52 isoform, has been identified as a primary signaling partner for the tyrosine(s)-phosphorylated cytoplasmic tails of activated ? 3 integrins. Inspired by our recent structure of the Shc PTB domain in complex with a bi-phosphorylated peptide derived from ? 3 cytoplasmic tail, we have initiated the investigation of Shc interaction with phospholipids of the membrane. We are particularly focused on PtdIns and their effects on Shc mediated integrin sign...
Lin, Yong-Chin; Chen, Jiann-Chu; Chen, Yu-Yuan; Liu, Chun-Hung; Cheng, Winton; Hsu, Chih-Hung; Tsui, Wen-Ching
2013-06-01
The full sequence of white shrimp Litopenaeus vannamei integrin β (LV-B) is 2879bp which encodes 787 amino acids (aa) of the open reading frame (ORF). The mature protein (764 aa) contains (1) an extracellular domain (ED) of 692 aa, (2) a transmembrane domain (TD) of 23 aa, and (3) a cytoplasmic domain (CD) of 49 aa. The cloned LV-B grouped together with crayfish Pacifastacus leniusculus integrin β (PL-B1), but was far away from vertebrate integrin β1, β3, β5, β6, β7, and β8, and another L. vannamei integrin β (LV). A Southern blot analysis indicated that the cloned LV-B was a single copy of genomic DNA. LV-B mRNA was expressed in all tissues, and was highly expressed in haemocytes. LV-B was downregulated in shrimp 24 and 96h after having received white spot syndrome virus (WSSV). LV-B expression by haemocytes of shrimp was higher in the postmoult (A and B) stage, and lower in the premoult (D2/D3) stage. LV-B expression was significantly higher by shrimp reared in 2.5‰ and 5‰ salinities. Shrimp injected with integrin β dsRNA showed gene silencing of integrin β after 36h. LV-B-silenced shrimp showed decreased hyaline cells (HCs), granular cells (GCs, including semi-granular cells), the total haemocyte count (THC), respiratory bursts (RBs), and lysozyme activity, but showed increased RB/HC, superoxide dismutase (SOD) activity/HC, and the phenoloxidase (PO) activity/GC. LV-B-silenced shrimp showed upregulated expressions of lipopolysaccharide- and β-glucan-binding protein (LGBP), peroxinectin (PX), prophenoloxidase I (proPO I), proPO II, proPO-activating enzyme (ppA), α2-macroglobulin (α2-M), cytMnSOD, mtMnSOD, and heat shock protein 70 (HSP70). It was concluded that integrin β plays important roles in proPO activation, phagocytosis, and the antioxidant system for immunomodulation in shrimp. Copyright © 2013 Elsevier Ltd. All rights reserved.
Effects of NR1 splicing on NR1/NR3B-type excitatory glycine receptors
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Orth Angela
2009-04-01
Full Text Available Abstract Background N-methyl-D-aspartate receptors (NMDARs are the most complex of ionotropic glutamate receptors (iGluRs. Subunits of this subfamily assemble into heteromers, which – depending on the subunit combination – may display very different pharmacological and electrophysiological properties. The least studied members of the NMDAR family, the NR3 subunits, have been reported to assemble with NR1 to form excitatory glycine receptors in heterologous expression systems. The heterogeneity of NMDARs in vivo is in part conferred to the receptors by splicing of the NR1 subunit, especially with regard to proton sensitivity. Results Here, we have investigated whether the NR3B subunit is capable of assembly with each of the eight functional NR1 splice variants, and whether the resulting receptors share the unique functional properties described for NR1-1a/NR3. We provide evidence that functional excitatory glycine receptors formed regardless of the NR1 isoform, and their pharmacological profile matched the one reported for NR1-1a/NR3: glycine alone fully activated the receptors, which were insensitive to glutamate and block by Mg2+. Surprisingly, amplitudes of agonist-induced currents showed little dependency on the C-terminally spliced NR1 variants in NR1/NR3B diheteromers. Even more strikingly, NR3B conferred proton sensitivity also to receptors containing NR1b variants – possibly via disturbing the "proton shield" of NR1b splice variants. Conclusion While functional assembly could be demonstrated for all combinations, not all of the specific interactions seen for NR1 isoforms with coexpressed NR2 subunits could be corroborated for NR1 assembly with NR3. Rather, NR3 abates trafficking effects mediated by the NR1 C terminus as well as the N-terminally mediated proton insensitivity. Thus, this study establishes that NR3B overrides important NR1 splice variant-specific receptor properties in NR1/NR3B excitatory glycine receptors.
Integrin β1, osmosensing, and chemoresistance in mouse ehrlich carcinoma cells
DEFF Research Database (Denmark)
Sørensen, Belinda Halling; Rasmussen, Line Jee Hartmann; Broberg, Bjørn Sindballe
2015-01-01
BACKGROUND/AIMS: Altered expression of the integrin family of cell adhesion receptors has been associated with initiation, progression, and metastasis of solid tumors as well as in the development of chemoresistance. Here, we investigated the role of integrins, in particular integrin β1, in cell ...
Fibronectin-bound α5β1 integrins sense load and signal to reinforce adhesion in less than a second
Strohmeyer, Nico; Bharadwaj, Mitasha; Costell, Mercedes; Fässler, Reinhard; Müller, Daniel J.
2017-12-01
Integrin-mediated mechanosensing of the extracellular environment allows cells to control adhesion and signalling. Whether cells sense and respond to force immediately upon ligand-binding is unknown. Here, we report that during adhesion initiation, fibroblasts respond to mechanical load by strengthening integrin-mediated adhesion to fibronectin (FN) in a biphasic manner. In the first phase, which depends on talin and kindlin as well as on the actin nucleators Arp2/3 and mDia, FN-engaged α5β1 integrins activate focal adhesion kinase (FAK) and c-Src in less than 0.5 s to steeply strengthen α5β1- and αV-class integrin-mediated adhesion. When the mechanical load exceeds a certain threshold, fibroblasts decrease adhesion and initiate the second phase, which is characterized by less steep adhesion strengthening. This unique, biphasic cellular adhesion response is mediated by α5β1 integrins, which form catch bonds with FN and signal to FN-binding integrins to reinforce cell adhesion much before visible adhesion clusters are formed.
DEFF Research Database (Denmark)
Bergmeier, W; Bouvard, D; Eble, J A
2001-01-01
Although alpha(2)beta(1) integrin (glycoprotein Ia/IIa) has been established as a platelet collagen receptor, its role in collagen-induced platelet activation has been controversial. Recently, it has been demonstrated that rhodocytin (also termed aggretin), a snake venom toxin purified from the v...
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Claudia S Priglinger
Full Text Available Proliferative vitreoretinopathy (PVR is a blinding disease frequently occurring after retinal detachment surgery. Adhesion, migration and matrix remodeling of dedifferentiated retinal pigment epithelial (RPE cells characterize the onset of the disease. Treatment options are still restrained and identification of factors responsible for the abnormal behavior of the RPE cells will facilitate the development of novel therapeutics. Galectin-3, a carbohydrate-binding protein, was previously found to inhibit attachment and spreading of retinal pigment epithelial cells, and thus bares the potential to counteract PVR-associated cellular events. However, the identities of the corresponding cell surface glycoprotein receptor proteins on RPE cells are not known. Here we characterize RPE-specific Gal-3 containing glycoprotein complexes using a proteomic approach. Integrin-β1, integrin-α3 and CD147/EMMPRIN, a transmembrane glycoprotein implicated in regulating matrix metalloproteinase induction, were identified as potential Gal-3 interactors on RPE cell surfaces. In reciprocal immunoprecipitation experiments we confirmed that Gal-3 associated with CD147 and integrin-β1, but not with integrin-α3. Additionally, association of Gal-3 with CD147 and integrin-β1 was observed in co-localization analyses, while integrin-α3 only partially co-localized with Gal-3. Blocking of CD147 and integrin-β1 on RPE cell surfaces inhibited binding of Gal-3, whereas blocking of integrin-α3 failed to do so, suggesting that integrin-α3 is rather an indirect interactor. Importantly, Gal-3 binding promoted pronounced clustering and co-localization of CD147 and integrin-β1, with only partial association of integrin-α3. Finally, we show that RPE derived CD147 and integrin-β1, but not integrin-α3, carry predominantly β-1,6-N-actyl-D-glucosamine-branched glycans, which are high-affinity ligands for Gal-3. We conclude from these data that extracellular Gal-3 triggers
Priglinger, Claudia S.; Szober, Christoph M.; Priglinger, Siegfried G.; Merl, Juliane; Euler, Kerstin N.; Kernt, Marcus; Gondi, Gabor; Behler, Jennifer; Geerlof, Arie; Kampik, Anselm; Ueffing, Marius; Hauck, Stefanie M.
2013-01-01
Proliferative vitreoretinopathy (PVR) is a blinding disease frequently occurring after retinal detachment surgery. Adhesion, migration and matrix remodeling of dedifferentiated retinal pigment epithelial (RPE) cells characterize the onset of the disease. Treatment options are still restrained and identification of factors responsible for the abnormal behavior of the RPE cells will facilitate the development of novel therapeutics. Galectin-3, a carbohydrate-binding protein, was previously found to inhibit attachment and spreading of retinal pigment epithelial cells, and thus bares the potential to counteract PVR-associated cellular events. However, the identities of the corresponding cell surface glycoprotein receptor proteins on RPE cells are not known. Here we characterize RPE-specific Gal-3 containing glycoprotein complexes using a proteomic approach. Integrin-β1, integrin-α3 and CD147/EMMPRIN, a transmembrane glycoprotein implicated in regulating matrix metalloproteinase induction, were identified as potential Gal-3 interactors on RPE cell surfaces. In reciprocal immunoprecipitation experiments we confirmed that Gal-3 associated with CD147 and integrin-β1, but not with integrin-α3. Additionally, association of Gal-3 with CD147 and integrin-β1 was observed in co-localization analyses, while integrin-α3 only partially co-localized with Gal-3. Blocking of CD147 and integrin-β1 on RPE cell surfaces inhibited binding of Gal-3, whereas blocking of integrin-α3 failed to do so, suggesting that integrin-α3 is rather an indirect interactor. Importantly, Gal-3 binding promoted pronounced clustering and co-localization of CD147 and integrin-β1, with only partial association of integrin-α3. Finally, we show that RPE derived CD147 and integrin-β1, but not integrin-α3, carry predominantly β-1,6-N-actyl-D-glucosamine-branched glycans, which are high-affinity ligands for Gal-3. We conclude from these data that extracellular Gal-3 triggers clustering of CD147 and
NOD1 cooperates with TLR2 to enhance T cell receptor-mediated activation in CD8 T cells.
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Blandine C Mercier
Full Text Available Pattern recognition receptors (PRR, like Toll-like receptors (TLR and NOD-like receptors (NLR, are involved in the detection of microbial infections and tissue damage by cells of the innate immune system. Recently, we and others have demonstrated that TLR2 can additionally function as a costimulatory receptor on CD8 T cells. Here, we establish that the intracytosolic receptor NOD1 is expressed and functional in CD8 T cells. We show that C12-iEDAP, a synthetic ligand for NOD1, has a direct impact on both murine and human CD8 T cells, increasing proliferation and effector functions of cells activated via their T cell receptor (TCR. This effect is dependent on the adaptor molecule RIP2 and is associated with an increased activation of the NF-κB, JNK and p38 signaling pathways. Furthermore, we demonstrate that NOD1 stimulation can cooperate with TLR2 engagement on CD8 T cells to enhance TCR-mediated activation. Altogether our results indicate that NOD1 might function as an alternative costimulatory receptor in CD8 T cells. Our study provides new insights into the function of NLR in T cells and extends to NOD1 the recent concept that PRR stimulation can directly control T cell functions.
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Bo Wu
2016-11-01
Full Text Available Hepatocellular carcinoma (HCC is currently the third most common cause of cancer-related death in the Asia-Pacific region. Our previous work showed that knockdown of CD98 significantly inhibits malignant HCC cell phenotypes in vitro and in vivo. The level of CD98 in the membrane is tightly regulated to mediate complex processes associated with cell–cell communication and intracellular signaling. In addition, the intracellular domain of CD98 (CD98-ICD seems to be of vital importance for recycling CD98 to the membrane after it is endocytosed. The intracellular and transmembrane domains of CD98 associate with β-integrins (primarily β1 but also β3, and this association is essential for CD98 mediation of integrin-like signaling and complements dominant suppression of β1-integrin. We speculated that isolated CD98-ICD would similarly suppress β1-integrin activation and inhibit the malignant behaviors of cancer cells. In particular, the exact role of CD98-ICD has not been studied independently in HCC. In this study, we found that ectopic expression of CD98-ICD inhibited the malignant phenotypes of HCC cells, and the mechanism possibly involves β1-integrin suppression. Moreover, the expression levels of CD98, β1-integrin-A (the activated form of β1-integrin and Ki-67 were significantly increased in HCC tissues relative to those of normal liver tissues. Therefore, our preliminary study indicates that ectopic CD98-ICD has an inhibitory role in the malignant development of HCC, and shows that CD98-ICD acts as a dominant negative mutant of CD98 that attenuates β1-integrin activation. CD98-ICD may emerge as a promising candidate for antitumor treatment.
Newman-Tancredi, Adrian; Cussac, Didier; Marini, Laetitia; Touzard, Manuelle; Millan, Mark J
2003-01-01
Serotonin 5-HT1B receptors couple to G-proteins of the Gi/o family. However, their activation of specific G-protein subtypes is poorly characterised. Using an innovative antibody capture/guanosine-5′-0-(3-[35S]thio)-triphosphate ([35S]GTPγS) binding strategy, we characterised Gαi3 subunit activation by h5-HT1B receptors stably expressed in Chinese hamster ovary (CHO) cells. The agonists, 5-HT, alniditan and BMS181,101, stimulated Gαi3, whereas methiothepin and SB224,289 behaved as inverse agonists. The selective 5-HT1B receptor ligand, S18127, modestly stimulated Gαi3 and reversed the actions of both 5-HT and methiothepin. S18127 (1 μM) also produced parallel, dextral shifts of the 5-HT and methiothepin isotherms. Isotopic dilution experiments ([35S]GTPγS versus GTPγS) revealed high-affinity [35S]GTPγS binding to Gαi3 subunits in the absence of receptor ligands indicating constitutive activity. High-affinity [35S]GTPγS binding was increased 2.8-fold by 5-HT with an increase in the affinity of GTPγS for Gαi3 subunits. In contrast, methiothepin halved the number of high-affinity binding sites and decreased their affinity. h5-HT1B receptor-mediated Gαi3 subunit activation was dependent on the concentration of NaCl. At 300 mM, 5-HT stimulated [35S]GTPγS binding, basal Gαi3 activation was low and methiothepin was inactive. In contrast, at 10 mM NaCl, basal activity was enhanced and the inverse agonist activity of methiothepin was accentuated. Under these conditions, 5-HT decreased Gαi3 activation. In conclusion, at h5-HT1B receptors expressed in CHO cells: (i) inverse agonist induced inhibition of Gαi3, and its reversal by S18127, reveals constitutive activation of this Gα subunit; (ii) constitutive Gαi3 activation can be quantified by isotopic dilution [35S]GTPγS binding and (iii) decreasing NaCl concentrations enhances Gαi3 activation and leads to protean agonist properties of 5-HT: that is a switch to inhibition of Gαi3. PMID:12684263
Phospho-Tyrosine(s) vs. Phosphatidylinositol Binding in Shc Mediated Integrin Signaling.
Lin, Xiaochen; Vinogradova, Olga
2015-04-01
The Shc adaptor protein, particularly its p52 isoform, has been identified as a primary signaling partner for the tyrosine(s)-phosphorylated cytoplasmic tails of activated β 3 integrins. Inspired by our recent structure of the Shc PTB domain in complex with a bi-phosphorylated peptide derived from β 3 cytoplasmic tail, we have initiated the investigation of Shc interaction with phospholipids of the membrane. We are particularly focused on PtdIns and their effects on Shc mediated integrin signaling in vitro . Here we present thermodynamic profiles and molecular details of the interactions between Shc, integrin, and PtdIns, all of which have been studied by ITC and solution NMR methods. A model of p52 Shc interaction with phosphorylated β 3 integrin cytoplasmic tail at the cytosolic face of the plasma membrane is proposed based on these data.
DEFF Research Database (Denmark)
Brockdorff, J; Kanner, S B; Nielsen, M
1998-01-01
beta2 integrin molecules are involved in a multitude of cellular events, including adhesion, migration, and cellular activation. Here, we studied the influence of beta2 integrins on interleukin-2 (IL-2)-mediated signal transduction in human CD4(+) T cell lines obtained from healthy donors...
Takawale, Abhijit; Zhang, Pu; Patel, Vaibhav B; Wang, Xiuhua; Oudit, Gavin; Kassiri, Zamaneh
2017-06-01
Myocardial fibrosis is excess accumulation of the extracellular matrix fibrillar collagens. Fibrosis is a key feature of various cardiomyopathies and compromises cardiac systolic and diastolic performance. TIMP1 (tissue inhibitor of metalloproteinase-1) is consistently upregulated in myocardial fibrosis and is used as a marker of fibrosis. However, it remains to be determined whether TIMP1 promotes tissue fibrosis by inhibiting extracellular matrix degradation by matrix metalloproteinases or via an matrix metalloproteinase-independent pathway. We examined the function of TIMP1 in myocardial fibrosis using Timp1 -deficient mice and 2 in vivo models of myocardial fibrosis (angiotensin II infusion and cardiac pressure overload), in vitro analysis of adult cardiac fibroblasts, and fibrotic myocardium from patients with dilated cardiomyopathy (DCM). Timp1 deficiency significantly reduced myocardial fibrosis in both in vivo models of cardiomyopathy. We identified a novel mechanism for TIMP1 action whereby, independent from its matrix metalloproteinase-inhibitory function, it mediates an association between CD63 (cell surface receptor for TIMP1) and integrin β1 on cardiac fibroblasts, initiates activation and nuclear translocation of Smad2/3 and β-catenin, leading to de novo collagen synthesis. This mechanism was consistently observed in vivo, in cultured cardiac fibroblasts, and in human fibrotic myocardium. In addition, after long-term pressure overload, Timp1 deficiency persistently reduced myocardial fibrosis and ameliorated diastolic dysfunction. This study defines a novel matrix metalloproteinase-independent function of TIMP1 in promoting myocardial fibrosis. As such targeting TIMP1 could prove to be a valuable approach in developing antifibrosis therapies. © 2017 American Heart Association, Inc.
DEFF Research Database (Denmark)
Christensen, Jan Pravsgaard; Marker, O; Thomsen, Allan Randrup
1996-01-01
The T-cell response to lymphocytic choriomeningitis virus was studied in mice with deficient expression of beta2-integrins or ICAM-1. In such mice, the generation of virus-specific cytotoxic T lymphocytes was only slightly impaired and bystander activation was as extensive as that observed in wild-type...... mice. T-cell-mediated inflammation, assessed as primary footpad swelling and susceptibility to intracerebral infection, was slightly compromised only in beta2-integrin-deficient mice. However, adoptive immunization of mutant mice soon after local infection did reveal a reduced capacity to support...... the inflammatory reaction, indicating that under conditions of more limited immune activation both molecules do play a role in formation of the inflammatory exudate. Finally, virus control was found to be somewhat impaired in both mutant strains. In conclusion, our results indicate that although LFA-1-ICAM-1...
Pakula, Rachel; Melchior, Aurélie; Denys, Agnès; Vanpouille, Christophe; Mazurier, Joël; Allain, Fabrice
2007-05-01
Many of the biological functions attributed to cell surface proteoglycans are dependent on the interaction with extracellular mediators through their heparan sulphate (HS) moieties and the participation of their core proteins in signaling events. A class of recently identified inflammatory mediators is secreted cyclophilins, which are mostly known as cyclosporin A-binding proteins. We previously demonstrated that cyclophilin B (CyPB) triggers chemotaxis and integrin-mediated adhesion of T lymphocytes mainly of the CD4+/CD45RO+ phenotype. These activities are related to interactions with two types of binding sites, CD147 and cell surface HS. Here, we demonstrate that CyPB-mediated adhesion of CD4+/CD45RO+ T cells is related to p44/42 mitogen-activated protein kinase (MAPK) activation by a mechanism involving CD147 and HS proteoglycans (HSPG). Although HSPG core proteins are represented by syndecan-1, -2, -4, CD44v3 and betaglycan in CD4+/CD45RO+ T cells, we found that only syndecan-1 is physically associated with CD147. The intensity of the heterocomplex increased in response to CyPB, suggesting a transient enhancement and/or stabilization in the association of CD147 to syndecan-1. Pretreatment with anti-syndecan-1 antibodies or knockdown of syndecan-1 expression by RNA interference dramatically reduced CyPB-induced p44/p42 MAPK activation and consequent migration and adhesion, supporting the model in which syndecan-1 serves as a binding subunit to form the fully active receptor of CyPB. Altogether, our findings provide a novel example of a soluble mediator in which a member of the syndecan family plays a critical role in efficient interaction with signaling receptors and initiation of cellular responses.
Pellegrini, Paul
This thesis reports on the synthesis, characterisation and biological evaluation of a number of metal complexes designed to interact with the alphavbeta3 integrin receptor, an important biological target that is heavily involved in angiogenesis, and thus cancer related processes. Two approaches were used to synthesise the integrin-avid targets. The first was to attach a variety of bifunctional chelators (BFC's) for the incorporation of different metal centres to a known integrin antagonist, L-748,415, developed by Merck. The BFC's used were the hydrazinonicotinamide (HYNIC) and monoamine monoamide dithiol (MAMA) systems for coordination to Tc-99m and rhenium of which was used as a characterization surrogate for the unstable Tc core. The 1,4,7,10-tetraazacyclotridecanetetraacetic acid (TRITA) BFC was attached for the inclusion of copper and lutetium. This 'conjugate' approach was designed to yield information on how the BFC and the linker length would affect the affinity for the integrin receptor. The second approach was an 'integrated' method where the chelation moiety was integral to the biologically relevant part of the molecule, which in the case of the alphavbeta3 integrin receptor, is the arginine-glycine-aspartic acid (RGD) mimicking sequence. Two complexes were created with a modified MAMA derivative placed between a benzimidazole moiety (arginine mimick) and the aspartic acid mimicking terminal carboxylic acid to see how it would affect binding while keeping the molecular weight relatively low. The molecules were tested in vitro against purified human alphavbeta3 integrin receptor protein in a solid phase receptor binding assay to evaluate their inhibition constants against a molecule of known high affinity and selectivity in [I125]L-775,219, the I125 labelled alphavbeta3 integrin antagonist. The radiolabelled analogues were also tested in vivo against the A375 human melanoma cell line transplanted into balb/c nude mice as well as Fischer rats implanted
Kim, A-Ram; Kim, Hyuk Soon; Lee, Jeong Min; Choi, Jung Ho; Kim, Se Na; Kim, Do Kyun; Kim, Ji Hyung; Mun, Se Hwan; Kim, Jie Wan; Jeon, Hyun Soo; Kim, Young Mi; Choi, Wahn Soo
2012-05-05
Osteoclasts, multinucleated bone-resorbing cells, are closely associated with bone diseases such as rheumatoid arthritis and osteoporosis. Osteoclasts are derived from hematopoietic precursor cells, and their differentiation is mediated by two cytokines, including macrophage colony stimulating factor and receptor activator of nuclear factor κB ligand (RANKL). Previous studies have shown that arctigenin exhibits an anti-inflammatory effect. However, the effect of arctigenin on osteoclast differentiation is yet to be elucidated. In this study, we found that arctigenin inhibited RANKL-mediated osteoclast differentiation in bone marrow macrophages in a dose-dependent manner and suppressed RANKL-mediated bone resorption. Additionally, the expression of typical marker proteins, such as NFATc1, c-Fos, TRAF6, c-Src, and cathepsin K, were significantly inhibited. Arctigenin inhibited the phosphorylation of Erk1/2, but not p38 and JNK, in a dose-dependent manner. Arctigenin also dramatically suppressed immunoreceptor tyrosine-based activation motif-mediated costimulatory signaling molecules, including Syk and PLCγ2, and Gab2. Notably, arctigenin inhibited the activation of Syk through RANKL stimulation. Furthermore, arctigenin prevented osteoclast differentiation in the calvarial bone of mice following stimulation with lipopolysaccharide. Our results show that arctigenin inhibits osteoclast differentiation in vitro and in vivo. Therefore, arctigenin may be useful for treating rheumatoid arthritis and osteoporosis. Copyright © 2012 Elsevier B.V. All rights reserved.
Stronger Dopamine D1 Receptor-Mediated Neurotransmission in Dyskinesia.
Farré, Daniel; Muñoz, Ana; Moreno, Estefanía; Reyes-Resina, Irene; Canet-Pons, Júlia; Dopeso-Reyes, Iria G; Rico, Alberto J; Lluís, Carme; Mallol, Josefa; Navarro, Gemma; Canela, Enric I; Cortés, Antonio; Labandeira-García, José L; Casadó, Vicent; Lanciego, José L; Franco, Rafael
2015-12-01
Radioligand binding assays to rat striatal dopamine D1 receptors showed that brain lateralization of the dopaminergic system were not due to changes in expression but in agonist affinity. D1 receptor-mediated striatal imbalance resulted from a significantly higher agonist affinity in the left striatum. D1 receptors heteromerize with dopamine D3 receptors, which are considered therapeutic targets for dyskinesia in parkinsonian patients. Expression of both D3 and D1-D3 receptor heteromers were increased in samples from 6-hydroxy-dopamine-hemilesioned rats rendered dyskinetic by treatment with 3, 4-dihydroxyphenyl-L-alanine (L-DOPA). Similar findings were obtained using striatal samples from primates. Radioligand binding studies in the presence of a D3 agonist led in dyskinetic, but not in lesioned or L-DOPA-treated rats, to a higher dopamine sensitivity. Upon D3-receptor activation, the affinity of agonists for binding to the right striatal D1 receptor increased. Excess dopamine coming from L-DOPA medication likely activates D3 receptors thus making right and left striatal D1 receptors equally responsive to dopamine. These results show that dyskinesia occurs concurrently with a right/left striatal balance in D1 receptor-mediated neurotransmission.
DEFF Research Database (Denmark)
Kermanizadeh, Ali; Villadsen, Klaus; Østrem, Ragnhild Garborg
2017-01-01
constructed with the aim of targeting integrins (i.e. vitronectin and/or fibronectin receptors) on activated endothelial cells. The peptide-conjugated liposomes induced only cytotoxicity at the highest concentration in non-activated or activated endothelial cells, as well as in co-culture of endothelial cells...... and macrophages. There was unaltered secretion of cytokines following exposure of peptide-conjugated liposomes to endothelial cells, indicating that the materials were not inflammogenic. Liposomes with a peptide targeting the fibronectin receptor (integrin α5β1) were more effective in targeting of activated....... Therefore, this study demonstrates the feasibility of constructing a peptide-conjugated cationic liposome, which displays targeting to activated endothelial cells at concentrations that are not cytotoxic or inflammogenic to the cells....
Syndecan-4 Phosphorylation Is a Control Point for Integrin Recycling
Morgan, Mark R.; Hamidi, Hellyeh; Bass, Mark D.; Warwood, Stacey; Ballestrem, Christoph; Humphries, Martin J.
2013-01-01
Summary Precise spatiotemporal coordination of integrin adhesion complex dynamics is essential for efficient cell migration. For cells adherent to fibronectin, differential engagement of α5β1 and αVβ3 integrins is used to elicit changes in adhesion complex stability, mechanosensation, matrix assembly, and migration, but the mechanisms responsible for receptor regulation have remained largely obscure. We identify phosphorylation of the membrane-intercalated proteoglycan syndecan-4 as an essential switch controlling integrin recycling. Src phosphorylates syndecan-4 and, by driving syntenin binding, leads to suppression of Arf6 activity and recycling of αVβ3 to the plasma membrane at the expense of α5β1. The resultant elevation in αVβ3 engagement promotes stabilization of focal adhesions. Conversely, abrogation of syndecan-4 phosphorylation drives surface expression of α5β1, destabilizes adhesion complexes, and disrupts cell migration. These data identify the dynamic spatiotemporal regulation of Src-mediated syndecan-4 phosphorylation as an essential switch controlling integrin trafficking and adhesion dynamics to promote efficient cell migration. PMID:23453597
Cardiac integrins the ties that bind.
Simpson, D G; Reaves, T A; Shih, D T; Burgess, W; Borg, T K; Terracio, L
1998-01-01
An elaborate series of morphogenetic events must be precisely coordinated during development to promote the formation of the elaborate three-dimensional structure of the normal heart. In this study we focus on discussing how interconnections between the cardiac myocyte and its surrounding environment regulate cardiac form and function. In vitro experiments from our laboratories provide direct evidence that cardiac cell shape is regulated by a dynamic interaction between constituents of the extracellular matrix (ECM) and by specific members of the integrin family of matrix receptors. Our data indicates that phenotypic information is stored in the tertiary structure and chemical identity of the ECM. This information appears to be actively communicated and transduced by the α1β1 integrin molecule into an intracellular signal that regulates cardiac cell shape and myofibrillar organization. In this study we have assessed the phenotypic consequences of suppressing the expression and accumulation of the α1 integrin molecule in aligned cultures of cardiac myocytes. In related experiments we have examined how the overexpression of α2 and α5 integrin, integrins normally not present or present at very low copy number on the cell surface of neonatal cardiac myocytes, affect cardiac protein metabolism. We also consider how biochemical signals and the mechanical signals mediated by the integrins may converge on common intracellular signaling pathways in the heart. Experiments with the whole embryo culture system indicate that angiotensin II, a peptide that carries information concerning cardiac load, plays a role in controling cardiac looping and the proliferation of myofibrils during development.
International Nuclear Information System (INIS)
Egami, Takuya; Ohuchida, Kenoki; Yasui, Takaharu; Onimaru, Manabu; Toma, Hiroki; Sato, Norihiro; Tanaka, Masao; Mizumoto, Kazuhiro; Matsumoto, Kunio
2009-01-01
Adenovirus-mediated gene therapy is a promising approach for the treatment of pancreatic cancer. We previously reported that radiation enhanced adenovirus-mediated gene expression in pancreatic cancer, suggesting that adenoviral gene therapy might be more effective in radioresistant pancreatic cancer cells. In the present study, we compared the transduction efficiency of adenovirus-delivered genes in radiosensitive and radioresistant cells, and investigated the underlying mechanisms. We used an adenovirus expressing the hepatocyte growth factor antagonist, NK4 (Ad-NK4), as a representative gene therapy. We established two radioresistant human pancreatic cancer cell lines using fractionated irradiation. Radiosensitive and radioresistant pancreatic cancer cells were infected with Ad-NK4, and NK4 levels in the cells were measured. In order to investigate the mechanisms responsible for the differences in the transduction efficiency between these cells, we measured expression of the genes mediating adenovirus infection and endocytosis. The results revealed that NK4 levels in radioresistant cells were significantly lower (P<0.01) than those in radiosensitive cells, although there were no significant differences in adenovirus uptake between radiosensitive cells and radioresistant cells. Integrin β3 was up-regulated and the Coxsackie virus and adenovirus receptor was down-regulated in radioresistant cells, and inhibition of integrin β3 promoted adenovirus gene transfer. These results suggest that inhibition of integrin β3 in radioresistant pancreatic cancer cells could enhance adenovirus-mediated gene therapy. (author)
Directory of Open Access Journals (Sweden)
ANA MARIA AVALOS
2002-01-01
Full Text Available Thy-1 is an abundant neuronal glycoprotein in mammals. Despite such prevalence, Thy-1 function remains largely obscure in the absence of a defined ligand. Recently described evidence that Thy-1 interacts with ß3 integrin on astrocytes will be discussed. Thy-1 binding to ß3 integrin triggers tyrosine phosphorylation of focal adhesion proteins in astrocytes, thereby promoting focal adhesion formation, cell attachment and spreading. Thy-1 has been reported to modulate neurite outgrowth by triggering a cellular response in neurons. However, our data indicate that Thy-1 can also initiate signaling events that promote adhesion of adjacent astrocytes to the underlying surface. Preliminary results suggest that morphological changes observed in the actin cytoskeleton of astrocytes as a consequence of Thy-1 binding is mediated by small GTPases from the Rho family. Our findings argue that Thy-1 functions in a bimodal fashion, as a receptor on neuronal cells and as a ligand for ß3 integrin receptor on astrocytes. Since Thy-1 is implicated in the inhibition of neurite outgrowth, signaling events in astrocytes are likely to play an important role in this process
Energy Technology Data Exchange (ETDEWEB)
Say, R Latin-Small-Letter-Dotless-I dvan, E-mail: rsay@anadolu.edu.tr [Anadolu Universitesi, Kimya Boeluemue, Fen Fakueltesi (Turkey); Yazar, Suzan [Sanovel Pharmaceutical Company (Turkey); Ugur, Alper; Huer, Deniz [Anadolu Universitesi, Kimya Boeluemue, Fen Fakueltesi (Turkey); Denizli, Adil [Hacettepe University, Department of Chemistry (Turkey); Ersoez, Arzu [Anadolu Universitesi, Kimya Boeluemue, Fen Fakueltesi (Turkey)
2013-01-15
Integrin family members are the main mediators of cell adhesion to the extracellular matrix and active as intra- and extracellular signaling molecules in a variety of processes. They bind to their ligands by interacting with short amino acid sequences, that is, RGD (arginine-glycine-aspartic acid) sequence. RGD sequences have been used to enhance cell binding to artificial surfaces, so RGD mimics have been used to block integrin binding to its ligand. Integrin-ligand interactions are dependent on divalent cations, and Mg{sup 2+} provide higher-affinity binding to ligand for many integrins. In this study, we have designed new integrin antagonists using methacryloyl amidoaspartic acid (MAASP) monomer-conjugated silanized super paramagnetic iron oxide nanoparticles (SPIONs, the size of the nanoparticles was verified with an average size of 32.6 nm) and poly(MAASP-co-EDMA) shell-decorated silanized SPIONs. Several mechanisms have been proposed to describe uptake of modified SPIONs into the cells, including receptor-mediated endocytosis. Our aim is to bind these modified SPIONs to the integrin-mediated aspartic acid ends of MAASP monomers and block integrin binding to their ligand.
1,25-dihydroxyvitamin D3 impairs NF-κB activation in human naive B cells
International Nuclear Information System (INIS)
Geldmeyer-Hilt, Kerstin; Heine, Guido; Hartmann, Bjoern; Baumgrass, Ria; Radbruch, Andreas; Worm, Margitta
2011-01-01
Highlights: → In naive B cells, VDR activation by calcitriol results in reduced NF-κB p105 and p50 protein expression. → Ligating the VDR with calcitriol causes reduced nuclear translocation of NF-κB p65. → Reduced nuclear amount of p65 after calcitriol incubation results in reduced binding of p65 on the p105 promoter. → Thus, vitamin D receptor signaling may reduce or prevent activation of B cells and unwanted immune responses, e.g. in IgE dependent diseases such as allergic asthma. -- Abstract: 1α,25-dihydroxyvitamin D 3 (calcitriol), the bioactive metabolite of vitamin D, modulates the activation and inhibits IgE production of anti-CD40 and IL-4 stimulated human peripheral B cells. Engagement of CD40 results in NF-κB p50 activation, which is essential for the class switch to IgE. Herein, we investigated by which mechanism calcitriol modulates NF-κB mediated activation of human naive B cells. Naive B cells were predominantly targeted by calcitriol in comparison with memory B cells as shown by pronounced induction of the VDR target gene cyp24a1. Vitamin D receptor activation resulted in a strongly reduced p105/p50 protein and mRNA expression in human naive B cells. This effect is mediated by impaired nuclear translocation of p65 and consequently reduced binding of p65 to its binding site in the p105 promoter. Our data indicate that the vitamin D receptor reduces NF-κB activation by interference with NF-κB p65 and p105. Thus, the vitamin D receptor inhibits costimulatory signal transduction in naive B cells, namely by reducing CD40 signaling.
Blenau, Wolfgang; Balfanz, Sabine; Baumann, Arnd
2017-10-30
The catecholamines norepinephrine and epinephrine regulate important physiological functions in vertebrates. In insects; these neuroactive substances are functionally replaced by the phenolamines octopamine and tyramine. Phenolamines activate specific guanine nucleotide-binding (G) protein-coupled receptors (GPCRs). Type 1 tyramine receptors are better activated by tyramine than by octopamine. In contrast; type 2 tyramine receptors are almost exclusively activated by tyramine. Functionally; activation of type 1 tyramine receptors leads to a decrease in the intracellular concentration of cAMP ([cAMP] i ) whereas type 2 tyramine receptors can mediate Ca 2+ signals or both Ca 2+ signals and effects on [cAMP] i . Here; we report that the American cockroach ( Periplaneta americana ) expresses a second type 1 tyramine receptor (PeaTAR1B) in addition to PeaTAR1A (previously called PeaTYR1). When heterologously expressed in flpTM cells; activation of PeaTAR1B by tyramine leads to a concentration-dependent decrease in [cAMP] i . Its activity can be blocked by a series of established antagonists. The functional characterization of two type 1 tyramine receptors from P. americana ; PeaTAR1A and PeaTAR1B; which respond to tyramine by changing cAMP levels; is a major step towards understanding the actions of tyramine in cockroach physiology and behavior; particularly in comparison to the effects of octopamine.
Directory of Open Access Journals (Sweden)
Wolfgang Blenau
2017-10-01
Full Text Available The catecholamines norepinephrine and epinephrine regulate important physiological functions in vertebrates. In insects; these neuroactive substances are functionally replaced by the phenolamines octopamine and tyramine. Phenolamines activate specific guanine nucleotide-binding (G protein-coupled receptors (GPCRs. Type 1 tyramine receptors are better activated by tyramine than by octopamine. In contrast; type 2 tyramine receptors are almost exclusively activated by tyramine. Functionally; activation of type 1 tyramine receptors leads to a decrease in the intracellular concentration of cAMP ([cAMP]i whereas type 2 tyramine receptors can mediate Ca2+ signals or both Ca2+ signals and effects on [cAMP]i. Here; we report that the American cockroach (Periplaneta americana expresses a second type 1 tyramine receptor (PeaTAR1B in addition to PeaTAR1A (previously called PeaTYR1. When heterologously expressed in flpTM cells; activation of PeaTAR1B by tyramine leads to a concentration-dependent decrease in [cAMP]i. Its activity can be blocked by a series of established antagonists. The functional characterization of two type 1 tyramine receptors from P. americana; PeaTAR1A and PeaTAR1B; which respond to tyramine by changing cAMP levels; is a major step towards understanding the actions of tyramine in cockroach physiology and behavior; particularly in comparison to the effects of octopamine.
Invasin of Yersinia pseudotuberculosis activates human peripheral B cells.
Lundgren, E; Carballeira, N; Vazquez, R; Dubinina, E; Bränden, H; Persson, H; Wolf-Watz, H
1996-01-01
The Yersinia pseudotuberculosis cell surface-located protein invasin was found to promote binding between the pathogen and resting peripheral B cells via beta 1 integrin receptors (CD29). B cells responded by expressing several activation markers and by growing, In contrast, T cells did not react, although these cells express CD29. An isogenic invA mutant failed to activate B cells. The mutation could be complemented by providing the invA+ gene in trans. Purified invasin alone did not activat...
D'Ursi, Pasqualina; Orro, Alessandro; Morra, Giulia; Moscatelli, Marco; Trombetti, Gabriele; Milanesi, Luciano; Rovida, Ermanna
2015-01-01
Activated Protein C (APC) is a multifunctional serine protease, primarily known for its anticoagulant function in the coagulation system. Several studies have already elucidated its role in counteracting apoptosis and inflammation in cells, while significant effort is still ongoing for defining its involvement in sepsis. Earlier literature has shown that the antiseptic function of APC is mediated by its binding to leukocyte integrins, which is due to the presence of the integrin binding motif Arg-Gly-Asp at the N-terminus of the APC catalytic chain. Many natural mutants have been identified in patients with Protein C deficiency diagnosis including a variant of specificity pocket (Gly216Asp). In this work, we present a molecular model of the complex of APC with αVβ3 integrin obtained by protein-protein docking approach. A computational analysis of this variant is hereby presented, based on molecular dynamics and docking simulations, aiming at investigating the effects of the Gly216Asp mutation on the protein conformation and inferring its functional implications. Our study shows that such mutation is likely to impair the protease activity while preserving the overall protein fold. Moreover, superposition of the integrin binding motifs in wild-type and mutant forms suggests that the interaction with integrin can still occur and thus the mutant is likely to retain its antiseptic function related to the neutrophyl integrin binding. Therapeutic applications could result in this APC mutant which retains antiseptic function without anticoagulant side effects.
Chen, Buxin; Dores, Michael R.; Grimsey, Neil; Canto, Isabel; Barker, Breann L.; Trejo, JoAnn
2011-01-01
Signaling by protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is regulated by desensitization and internalization. PAR1 desensitization is mediated by β-arrestins, like most classic GPCRs. In contrast, internalization of PAR1 occurs through a clathrin- and dynamin-dependent pathway independent of β-arrestins. PAR1 displays two modes of internalization. Constitutive internalization of unactivated PAR1 is mediated by the clathrin adaptor protein complex-2 (AP-2), where the μ2-adaptin subunit binds directly to a tyrosine-based motif localized within the receptor C-tail domain. However, AP-2 depletion only partially inhibits agonist-induced internalization of PAR1, suggesting a function for other clathrin adaptors in this process. Here, we now report that AP-2 and epsin-1 are both critical mediators of agonist-stimulated PAR1 internalization. We show that ubiquitination of PAR1 and the ubiquitin-interacting motifs of epsin-1 are required for epsin-1-dependent internalization of activated PAR1. In addition, activation of PAR1 promotes epsin-1 de-ubiquitination, which may increase its endocytic adaptor activity to facilitate receptor internalization. AP-2 also regulates activated PAR1 internalization via recognition of distal C-tail phosphorylation sites rather than the canonical tyrosine-based motif. Thus, AP-2 and epsin-1 are both required to promote efficient internalization of activated PAR1 and recognize discrete receptor sorting signals. This study defines a new pathway for internalization of mammalian GPCRs. PMID:21965661
De, Pradip; Carlson, Jennifer H; Jepperson, Tyler; Willis, Scooter; Leyland-Jones, Brian; Dey, Nandini
2017-01-10
The acquisition of integrin-directed metastasis-associated (ID-MA) phenotypes by Triple-Negative Breast Cancer (TNBC) cells is caused by an upregulation of the Wnt-beta-catenin pathway (WP). We reported that WP is one of the salient genetic features of TNBC. RAC-GTPases, small G-proteins which transduce signals from cell surface proteins including integrins, have been implicated in tumorigenesis and metastasis by their role in essential cellular functions like motility. The collective percentage of alteration(s) in RAC1 in ER+ve BC was lower as compared to ER-ve BC (35% vs 57%) (brca/tcga/pub2015). High expression of RAC1 was associated with poor outcome for RFS with HR=1.48 [CI: 1.15-1.9] p=0.0019 in the Hungarian ER-veBC cohort. Here we examined how WP signals are transduced via RAC1 in the context of ID-MA phenotypes in TNBC. Using pharmacological agents (sulindac sulfide), genetic tools (beta-catenin siRNA), WP modulators (Wnt-C59, XAV939), RAC1 inhibitors (NSC23766, W56) and WP stimulations (LWnt3ACM, Wnt3A recombinant) in a panel of 6-7 TNBC cell lines, we studied fibronectin-directed (1) migration, (2) matrigel invasion, (3) RAC1 and Cdc42 activation, (4) actin dynamics (confocal microscopy) and (5) podia-parameters. An attenuation of WP, which (a) decreased cellular levels of beta-catenin, as well as its nuclear active-form, (b) decreased fibronectin-induced migration, (c) decreased invasion, (d) altered actin dynamics and (e) decreased podia-parameters was successful in blocking fibronectin-mediated RAC1/Cdc42 activity. Both Wnt-antagonists and RAC1 inhibitors blocked fibronectin-induced RAC1 activation and inhibited the fibronectin-induced ID-MA phenotypes following specific WP stimulation by LWnt3ACM as well as Wnt3A recombinant protein. To test a direct involvement of RAC1-activation in WP-mediated ID-MA phenotypes, we stimulated brain-metastasis specific MDA-MB231BR cells with LWnt3ACM. LWnt3ACM-stimulated fibronectin-directed migration was blocked by
Maldonado, H; Calderon, C; Burgos-Bravo, F; Kobler, O; Zuschratter, W; Ramirez, O; Härtel, S; Schneider, P; Quest, A F G; Herrera-Molina, R; Leyton, L
2017-02-01
Two key proteins for cellular communication between astrocytes and neurons are αvβ3 integrin and the receptor Thy-1. Binding of these molecules in the same (cis) or on adjacent (trans) cellular membranes induces Thy-1 clustering, triggering actin cytoskeleton remodeling. Molecular events that could explain how the Thy-1-αvβ3 integrin interaction signals have only been studied separately in different cell types, and the detailed transcellular communication and signal transduction pathways involved in neuronal cytoskeleton remodeling remain unresolved. Using biochemical and genetic approaches, single-molecule tracking, and high-resolution nanoscopy, we provide evidence that upon binding to αvβ3 integrin, Thy-1 mobility decreased while Thy-1 nanocluster size increased. This occurred concomitantly with inactivation and exclusion of the non-receptor tyrosine kinase Src from the Thy-1/C-terminal Src kinase (Csk)-binding protein (CBP)/Csk complex. The Src inactivation decreased the p190Rho GTPase activating protein phosphorylation, promoting RhoA activation, cofilin, and myosin light chain II phosphorylation and, consequently, neurite shortening. Finally, silencing the adaptor CBP demonstrated that this protein was a key transducer in the Thy-1 signaling cascade. In conclusion, these data support the hypothesis that the Thy-1-CBP-Csk-Src-RhoA-ROCK axis transmitted signals from astrocytic integrin-engaged Thy-1 (trans) to the neuronal actin cytoskeleton. Importantly, the β3 integrin in neurons (cis) was not found to be crucial for neurite shortening. This is the first study to detail the signaling pathway triggered by αvβ3, the endogenous Thy-1 ligand, highlighting the role of membrane-bound integrins as trans acting ligands in astrocyte-neuron communication. Copyright © 2016 Elsevier B.V. All rights reserved.
Plasma extravasation mediated by lipopolysaccharide-induction of kinin B1 receptors in rat tissues
Directory of Open Access Journals (Sweden)
Paulo Roberto Wille
2001-01-01
Full Text Available The present study was performed to: (a evaluate the effects of kinin B1 (Sar{D-Phe8}-des-Arg9-BK; 10 nmol/kg and B2 (bradykinin (BK; 10 nmol/kg receptor agonists on plasma extravasation in selected rat tissues; (b determine the contribution of a lipopolysaccharide (LPS (100 μ g/kg to the effects triggered by B1 and B2 agonists; and (c characterize the selectivity of B1 ({Leu8}desArg9-BK; 10 nmol/kg and B2 (HOE 140; 10 nmol/kg antagonists as inhibitors of this kinin-induced phenomenon. B1 and B2 agonists were shown to increase plasma extravasation in the duodenum, ileum and also in the urinary bladder of the rat. LPS pretreatment enhanced the plasma extravasation mediated only by the B1 agonist in the duodenum, ileum, trachea, main and segmentar bronchi. These effects were prevented by the B1. but not the B2 antagonist. In normal rats, the B2 antagonist inhibited the effect of B2 agonist in all the tissues analyzed. However, in LPS-treated rats, the B2 antagonist was ineffective in the urinary bladder.
Role of the hinge region of glucocorticoid receptor for HEXIM1-mediated transcriptional repression
International Nuclear Information System (INIS)
Yoshikawa, Noritada; Shimizu, Noriaki; Sano, Motoaki; Ohnuma, Kei; Iwata, Satoshi; Hosono, Osamu; Fukuda, Keiichi; Morimoto, Chikao
2008-01-01
We previously reported that HEXIM1 (hexamethylene bisacetamide-inducible protein 1), which suppresses transcription elongation via sequestration of positive transcription elongation factor b (P-TEFb) using 7SK RNA as a scaffold, directly associates with glucocorticoid receptor (GR) to suppress glucocorticoid-inducible gene activation. Here, we revealed that the hinge region of GR is essential for its interaction with HEXIM1, and that oxosteroid receptors including GR show sequence homology in their hinge region and interact with HEXIM1, whereas the other members of nuclear receptors do not. We also showed that HEXIM1 suppresses GR-mediated transcription in two ways: sequestration of P-TEFb by HEXIM1 and direct interaction between GR and HEXIM1. In contrast, peroxisome proliferator-activated receptor γ-dependent gene expression is negatively modulated by HEXIM1 solely via sequestration of P-TEFb. We, therefore, conclude that HEXIM1 may act as a gene-selective transcriptional regulator via direct interaction with certain transcriptional regulators including GR and contribute to fine-tuning of, for example, glucocorticoid-mediated biological responses
Surface receptor Toso controls B cell-mediated regulation of T cell immunity.
Yu, Jinbo; Duong, Vu Huy Hoang; Westphal, Katrin; Westphal, Andreas; Suwandi, Abdulhadi; Grassl, Guntram A; Brand, Korbinian; Chan, Andrew C; Föger, Niko; Lee, Kyeong-Hee
2018-05-01
The immune system is tightly controlled by regulatory processes that allow for the elimination of invading pathogens, while limiting immunopathological damage to the host. In the present study, we found that conditional deletion of the cell surface receptor Toso on B cells unexpectedly resulted in impaired proinflammatory T cell responses, which led to impaired immune protection in an acute viral infection model and was associated with reduced immunopathological tissue damage in a chronic inflammatory context. Toso exhibited its B cell-inherent immunoregulatory function by negatively controlling the pool of IL-10-competent B1 and B2 B cells, which were characterized by a high degree of self-reactivity and were shown to mediate immunosuppressive activity on inflammatory T cell responses in vivo. Our results indicate that Toso is involved in the differentiation/maintenance of regulatory B cells by fine-tuning B cell receptor activation thresholds. Furthermore, we showed that during influenza A-induced pulmonary inflammation, the application of Toso-specific antibodies selectively induced IL-10-competent B cells at the site of inflammation and resulted in decreased proinflammatory cytokine production by lung T cells. These findings suggest that Toso may serve as a novel therapeutic target to dampen pathogenic T cell responses via the modulation of IL-10-competent regulatory B cells.
Pak, Jhang Ho; Bashir, Qudsia; Kim, In Ki; Hong, Sung-Jong; Maeng, Sejung; Bahk, Young Yil; Kim, Tong-Soo
2017-06-01
Cholangiocarcinoma (CCA) is a slow-growing but highly metastatic cancer. Its metastatic potential largely explains its high mortality rate. A recognized risk factor for CCA development is infection with the liver flukes Opisthorchis viverrini and Clonorchis sinensis. We previously reported that the excretory-secretory products (ESPs) of C. sinensis promoted the three-dimensional aggregation and invasion of CCA cells. In the present study, a quantitative real-time PCR array of extracellular matrix (ECM) and adhesion molecules was used to examine the regulatory mechanism of ESP-mediated CCA cell migration and invasion. In particular, the expression levels of integrin α isoforms and β4 were upregulated in response to ESPs. Increased expression of integrin β4 was probably correlated with activation of focal adhesion kinase (FAK) and the steroid receptor coactivator (Src) family kinase and the subsequent activation of two downstream focal adhesion molecules, paxillin and vinculin. Moreover, inhibition of FAK/Src activation reduced paxillin and vinculin phosphorylation and attenuated ESP-induced CCA cell migration and invasion. These findings suggest that the integrin β4-FAK/Src signaling axis may play a crucial role in clonorchiasis-associated CCA metastasis during tumor progression. Copyright © 2017 Elsevier B.V. All rights reserved.
Fang, Xiao-Qian; Qiao, Haifa; Groveman, Bradley R; Feng, Shuang; Pflueger, Melissa; Xin, Wen-Kuan; Ali, Mohammad K; Lin, Shuang-Xiu; Xu, Jindong; Duclot, Florian; Kabbaj, Mohamed; Wang, Wei; Ding, Xin-Sheng; Santiago-Sim, Teresa; Jiang, Xing-Hong; Salter, Michael W; Yu, Xian-Min
2015-11-19
Constitutive and regulated internalization of cell surface proteins has been extensively investigated. The regulated internalization has been characterized as a principal mechanism for removing cell-surface receptors from the plasma membrane, and signaling to downstream targets of receptors. However, so far it is still not known whether the functional properties of remaining (non-internalized) receptor/channels may be regulated by internalization of the same class of receptor/channels. The N-methyl-D-aspartate receptor (NMDAR) is a principal subtype of glutamate-gated ion channel and plays key roles in neuronal plasticity and memory functions. NMDARs are well-known to undergo two types of regulated internalization - homologous and heterologous, which can be induced by high NMDA/glycine and DHPG, respectively. In the present work, we investigated effects of regulated NMDAR internalization on the activity of residual cell-surface NMDARs and neuronal functions. In electrophysiological experiments we discovered that the regulated internalization of NMDARs not only reduced the number of cell surface NMDARs but also caused an inhibition of the activity of remaining (non-internalized) surface NMDARs. In biochemical experiments we identified that this functional inhibition of remaining surface NMDARs was mediated by increased serine phosphorylation of surface NMDARs, resulting from the activation of protein kinase D1 (PKD1). Knockdown of PKD1 did not affect NMDAR internalization but prevented the phosphorylation and inhibition of remaining surface NMDARs and NMDAR-mediated synaptic functions. These data demonstrate a novel concept that regulated internalization of cell surface NMDARs not only reduces the number of NMDARs on the cell surface but also causes an inhibition of the activity of remaining surface NMDARs through intracellular signaling pathway(s). Furthermore, modulating the activity of remaining surface receptors may be an effective approach for treating receptor
Rac-mediated Stimulation of Phospholipase Cγ2 Amplifies B Cell Receptor-induced Calcium Signaling*♦
Walliser, Claudia; Tron, Kyrylo; Clauss, Karen; Gutman, Orit; Kobitski, Andrei Yu.; Retlich, Michael; Schade, Anja; Röcker, Carlheinz; Henis, Yoav I.; Nienhaus, G. Ulrich; Gierschik, Peter
2015-01-01
The Rho GTPase Rac is crucially involved in controlling multiple B cell functions, including those regulated by the B cell receptor (BCR) through increased cytosolic Ca2+. The underlying molecular mechanisms and their relevance to the functions of intact B cells have thus far remained unknown. We have previously shown that the activity of phospholipase Cγ2 (PLCγ2), a key constituent of the BCR signalosome, is stimulated by activated Rac through direct protein-protein interaction. Here, we use a Rac-resistant mutant of PLCγ2 to functionally reconstitute cultured PLCγ2-deficient DT40 B cells and to examine the effects of the Rac-PLCγ2 interaction on BCR-mediated changes of intracellular Ca2+ and regulation of Ca2+-regulated and nuclear-factor-of-activated-T-cell-regulated gene transcription at the level of single, intact B cells. The results show that the functional Rac-PLCγ2 interaction causes marked increases in the following: (i) sensitivity of B cells to BCR ligation; (ii) BCR-mediated Ca2+ release from intracellular stores; (iii) Ca2+ entry from the extracellular compartment; and (iv) nuclear translocation of the Ca2+-regulated nuclear factor of activated T cells. Hence, Rac-mediated stimulation of PLCγ2 activity serves to amplify B cell receptor-induced Ca2+ signaling. PMID:25903139
GABA-B receptor activation and conflict behavior
International Nuclear Information System (INIS)
Ketelaars, C.E.J.; Bollen, E.L.; Rigter, H.; Bruinvels, J.
1988-01-01
Baclofen and oxazepam enhance extinction of conflict behavior in the Geller-Seifter test while baclofen and diazepam release punished behavior in Vogel's conflict test. In order to investigate the possibility that the effect of the selective GABA-B receptor agonist baclofen is mediated indirectly via the GABA-A/benzodiazepine receptor complex, the effect of pretreatment of rats with baclofen on [ 3 H]-diazepam binding to washed and unwashed cortical and cerebellar membranes of rats has been studied. Baclofen pretreatment increase Bmax in washed cerebellar membranes when bicuculline was present in the incubation mixture. No effect was seen in cortical membranes. The present results render it unlikely that the effect of baclofen on extinction of conflict behavior and punished drinking is mediated via the GABA-A/benzodiazepine receptor complex. 50 references, 1 figure, 4 tables
Directory of Open Access Journals (Sweden)
Campos Maria M
2010-12-01
Full Text Available Abstract Background Kinin B1 receptors are inducible molecules up-regulated after inflammatory stimuli. This study evaluated the relevance of kinin B1 receptors in a mouse depression behavior model. Methods Mice were exposed to a 5-min swimming session, and 30 min later they were injected with E. coli lipopolysaccharide (LPS. Depression-like behavior was assessed by determining immobility time in a tail suspension test. Different brain structures were collected for molecular and immunohistochemical studies. Anhedonia was assessed by means of a sucrose intake test. Results Our protocol elicited an increase in depression-like behavior in CF1 mice, as assessed by the tail-suspension test, at 24 h. This behavior was significantly reduced by treatment with the selective B1 receptor antagonists R-715 and SSR240612. Administration of SSR240612 also prevented an increase in number of activated microglial cells in mouse hippocampus, but did not affect a reduction in expression of mRNA for brain-derived neurotrophic factor. The increased immobility time following LPS treatment was preceded by an enhancement of hippocampal and cortical B1 receptor mRNA expression (which were maximal at 1 h, and a marked production of TNFα in serum, brain and cerebrospinal fluid (between 1 and 6 h. The depression-like behavior was virtually abolished in TNFα p55 receptor-knockout mice, and increased B1 receptor mRNA expression was completely absent in this mouse strain. Furthermore, treatment with SSR240612 was also effective in preventing anhedonia in LPS-treated mice, as assessed using a sucrose preference test. Conclusion Our data show, for the first time, involvement of kinin B1 receptors in depressive behavioral responses, in a process likely associated with microglial activation and TNFα production. Thus, selective and orally active B1 receptor antagonists might well represent promising pharmacological tools for depression therapy.
1,25-dihydroxyvitamin D{sub 3} impairs NF-{kappa}B activation in human naive B cells
Energy Technology Data Exchange (ETDEWEB)
Geldmeyer-Hilt, Kerstin, E-mail: kerstin.hilt@charite.de [Allergie-Centrum-Charite, CCM, Klinik fuer Dermatologie und Allergologie, Charite - Universitaetsmedizin Berlin, Chariteplatz 1, 10117 Berlin (Germany); Heine, Guido, E-mail: guido.heine@charite.de [Allergie-Centrum-Charite, CCM, Klinik fuer Dermatologie und Allergologie, Charite - Universitaetsmedizin Berlin, Chariteplatz 1, 10117 Berlin (Germany); Deutsches Rheuma-Forschungszentrum Berlin, Chariteplatz 1, 10117 Berlin (Germany); Hartmann, Bjoern, E-mail: bjoern.hartmann@charite.de [Allergie-Centrum-Charite, CCM, Klinik fuer Dermatologie und Allergologie, Charite - Universitaetsmedizin Berlin, Chariteplatz 1, 10117 Berlin (Germany); Baumgrass, Ria, E-mail: baumgrass@drfz.de [Deutsches Rheuma-Forschungszentrum Berlin, Chariteplatz 1, 10117 Berlin (Germany); Radbruch, Andreas, E-mail: radbruch@drfz.de [Deutsches Rheuma-Forschungszentrum Berlin, Chariteplatz 1, 10117 Berlin (Germany); Worm, Margitta, E-mail: margitta.worm@charite.de [Allergie-Centrum-Charite, CCM, Klinik fuer Dermatologie und Allergologie, Charite - Universitaetsmedizin Berlin, Chariteplatz 1, 10117 Berlin (Germany)
2011-04-22
Highlights: {yields} In naive B cells, VDR activation by calcitriol results in reduced NF-{kappa}B p105 and p50 protein expression. {yields} Ligating the VDR with calcitriol causes reduced nuclear translocation of NF-{kappa}B p65. {yields} Reduced nuclear amount of p65 after calcitriol incubation results in reduced binding of p65 on the p105 promoter. {yields} Thus, vitamin D receptor signaling may reduce or prevent activation of B cells and unwanted immune responses, e.g. in IgE dependent diseases such as allergic asthma. -- Abstract: 1{alpha},25-dihydroxyvitamin D{sub 3} (calcitriol), the bioactive metabolite of vitamin D, modulates the activation and inhibits IgE production of anti-CD40 and IL-4 stimulated human peripheral B cells. Engagement of CD40 results in NF-{kappa}B p50 activation, which is essential for the class switch to IgE. Herein, we investigated by which mechanism calcitriol modulates NF-{kappa}B mediated activation of human naive B cells. Naive B cells were predominantly targeted by calcitriol in comparison with memory B cells as shown by pronounced induction of the VDR target gene cyp24a1. Vitamin D receptor activation resulted in a strongly reduced p105/p50 protein and mRNA expression in human naive B cells. This effect is mediated by impaired nuclear translocation of p65 and consequently reduced binding of p65 to its binding site in the p105 promoter. Our data indicate that the vitamin D receptor reduces NF-{kappa}B activation by interference with NF-{kappa}B p65 and p105. Thus, the vitamin D receptor inhibits costimulatory signal transduction in naive B cells, namely by reducing CD40 signaling.
International Nuclear Information System (INIS)
Say, Rıdvan; Yazar, Suzan; Uğur, Alper; Hür, Deniz; Denizli, Adil; Ersöz, Arzu
2013-01-01
Integrin family members are the main mediators of cell adhesion to the extracellular matrix and active as intra- and extracellular signaling molecules in a variety of processes. They bind to their ligands by interacting with short amino acid sequences, that is, RGD (arginine-glycine-aspartic acid) sequence. RGD sequences have been used to enhance cell binding to artificial surfaces, so RGD mimics have been used to block integrin binding to its ligand. Integrin–ligand interactions are dependent on divalent cations, and Mg 2+ provide higher-affinity binding to ligand for many integrins. In this study, we have designed new integrin antagonists using methacryloyl amidoaspartic acid (MAASP) monomer-conjugated silanized super paramagnetic iron oxide nanoparticles (SPIONs, the size of the nanoparticles was verified with an average size of 32.6 nm) and poly(MAASP-co-EDMA) shell-decorated silanized SPIONs. Several mechanisms have been proposed to describe uptake of modified SPIONs into the cells, including receptor-mediated endocytosis. Our aim is to bind these modified SPIONs to the integrin-mediated aspartic acid ends of MAASP monomers and block integrin binding to their ligand.
Robles, Eloy F.; Mena-Varas, Maria; Barrio, Laura; Merino-Cortes, Sara V.; Balogh, Péter; Du, Ming-Qing; Akasaka, Takashi; Parker, Anton; Roa, Sergio; Panizo, Carlos; Martin-Guerrero, Idoia; Siebert, Reiner; Segura, Victor; Agirre, Xabier; Macri-Pellizeri, Laura; Aldaz, Beatriz; Vilas-Zornoza, Amaia; Zhang, Shaowei; Moody, Sarah; Calasanz, Maria Jose; Tousseyn, Thomas; Broccardo, Cyril; Brousset, Pierre; Campos-Sanchez, Elena; Cobaleda, Cesar; Sanchez-Garcia, Isidro; Fernandez-Luna, Jose Luis; Garcia-Muñoz, Ricardo; Pena, Esther; Bellosillo, Beatriz; Salar, Antonio; Baptista, Maria Joao; Hernandez-Rivas, Jesús Maria; Gonzalez, Marcos; Terol, Maria Jose; Climent, Joan; Ferrandez, Antonio; Sagaert, Xavier; Melnick, Ari M.; Prosper, Felipe; Oscier, David G.; Carrasco, Yolanda R.; Dyer, Martin J. S.; Martinez-Climent, Jose A.
2016-01-01
NKX2 homeobox family proteins have a role in cancer development. Here we show that NKX2-3 is overexpressed in tumour cells from a subset of patients with marginal-zone lymphomas, but not with other B-cell malignancies. While Nkx2-3-deficient mice exhibit the absence of marginal-zone B cells, transgenic mice with expression of NKX2-3 in B cells show marginal-zone expansion that leads to the development of tumours, faithfully recapitulating the principal clinical and biological features of human marginal-zone lymphomas. NKX2-3 induces B-cell receptor signalling by phosphorylating Lyn/Syk kinases, which in turn activate multiple integrins (LFA-1, VLA-4), adhesion molecules (ICAM-1, MadCAM-1) and the chemokine receptor CXCR4. These molecules enhance migration, polarization and homing of B cells to splenic and extranodal tissues, eventually driving malignant transformation through triggering NF-κB and PI3K-AKT pathways. This study implicates oncogenic NKX2-3 in lymphomagenesis, and provides a valid experimental mouse model for studying the biology and therapy of human marginal-zone B-cell lymphomas. PMID:27297662
Sigma-1 receptor enhances neurite elongation of cerebellar granule neurons via TrkB signaling.
Kimura, Yuriko; Fujita, Yuki; Shibata, Kumi; Mori, Megumi; Yamashita, Toshihide
2013-01-01
Sigma-1 receptor (Sig-1R) is an integral membrane protein predominantly expressed in the endoplasmic reticulum. Sig-1R demonstrates a high affinity to various synthetic compounds including well-known psychotherapeutic drugs in the central nervous system (CNS). For that, it is considered as an alternative target for psychotherapeutic drugs. On the cellular level, when Sig-1R is activated, it is known to play a role in neuroprotection and neurite elongation. These effects are suggested to be mediated by its ligand-operated molecular chaperone activity, and/or upregulation of various Ca(2+) signaling. In addition, recent studies show that Sig-1R activation induces neurite outgrowth via neurotrophin signaling. Here, we tested the hypothesis that Sig-1R activation promotes neurite elongation through activation of tropomyosin receptor kinase (Trk), a family of neurotrophin receptors. We found that 2-(4-morpholinethyl)1-phenylcyclohexanecarboxylate (PRE-084), a selective Sig-1R agonist, significantly promoted neurite outgrowth, and K252a, a Trk inhibitor, attenuated Sig-1R-mediated neurite elongation in cerebellar granule neurons (CGNs). Moreover, we revealed that Sig-1R interacts with TrkB, and PRE-084 treatment enhances phosphorylation of Y515, but not Y706. Thus, our results indicate that Sig-1R activation promotes neurite outgrowth in CGNs through Y515 phosphorylation of TrkB.
Directory of Open Access Journals (Sweden)
W. Barry Edwards
2009-03-01
Full Text Available Integrins, particularly the αvβ3 heterodimers, play important roles in tumor-induced angiogenesis and invasiveness. To image the expression pattern of the αvβ3 integrin in tumors through a multimodality imaging paradigm, we prepared a cyclic RGDyK peptide analogue (LS308 bearing a tetraazamacrocycle 1,4,7,10-tetraazacyclododecane-N, N′, N″, N‴-tetraacetic acid (DOTA and a lipophilic near-infrared (NIR fluorescent dye cypate. The αvβ3 integrin binding affinity and the internalization properties of LS308 mediated by the αvβ3 integrin in 4t1luc cells were investigated by receptor binding assay and fluorescence microscopy, respectively. The in vivo distribution of 111In-labeled LS308 in a 4t1luc tumor-bearing mouse model was studied by fluorescence, bioluminescence, planar gamma, and single-photon emission computed tomography (SPECT. The results show that LS308 has high affinity for αvβ3 integrin and internalized preferentially via the αvβ3 integrin-mediated endocytosis in 4t1luc cells. We also found that LS308 selectively accumulated in αvβ3-positve tumors in a receptor-specific manner and was visualized by the four imaging methods. Whereas the endogenous bioluminescence imaging identified the ensemble of the tumor tissue, the fluorescence and SPECT methods with the exogenous contrast agent LS308 reported the local expression of αvβ3 integrin. Thus, the multimodal imaging approach could provide important complementary diagnostic information for monitoring the efficacy of new antiangiogenic drugs.
International Nuclear Information System (INIS)
Schwarz, Margaret A.; Zheng, Hiahua; Liu, Jie; Corbett, Siobhan; Schwarz, Roderich E.
2005-01-01
Mature Endothelial-Monocyte Activating Polypeptide (mEMAP) II functions as a potent antiangiogenic peptide. Although the anti-tumor effect of mEMAP II has been described, little is known regarding its mechanism of action. Observations that mEMAP II induced apoptosis only in a subset of migrating and proliferating endothelial cells (EC) suggests a targeted effect on cells engaged in angiogenic activities which are known to rely upon cell adhesion and migration. Indeed, we demonstrate that mEMAP II inhibited fibronectin (FN) dependent microvascular EC (MEC) adhesion and spreading and we show that this depends upon the alpha5 beta1 integrin. Immunofluorescence analysis demonstrated that mEMAP II-dependent blockade of FN-alpha5 beta1 interactions was associated with disassembly of both actin stress fiber networks and FN matrix. These findings suggest that mEMAP II blocks MEC adhesion and spreading on fibronectin, via a direct interaction with the integrin alpha5 beta1, thus implicating that alpha5 integrin may be a mediator of mEMAP II's antiangiogenic function
Elucidating the role of select cytoplasmic proteins in altering diffusion of integrin receptors.
Sander, Suzanne; Arora, Neha; Smith, Emily A
2012-06-01
Cytoplasmic proteins that affect integrin diffusion in the cell membrane are identified using a combination of fluorescence recovery after photobleaching (FRAP) and RNA interference. Integrin receptors are essential for many cellular events, and alterations in lateral diffusion are one mechanism for modulating their function. In cells expressing native cytoplasmic protein concentrations and spread on a slide containing integrin extracellular ligand, 45 ± 2% of the integrin is mobile with a time-dependent 5.2 ± 0.9 × 10(-9) cm(2)/s diffusion coefficient at 1 s. The time exponent is 0.90 ± 0.07, indicating integrin diffusion moderately slows at longer times. The role of a specific cytoplasmic protein in altering integrin diffusion is revealed through changes in the FRAP curve after reducing the cytoplasmic protein's expression. Decreased expression of cytoplasmic proteins rhea, focal adhesion kinase (FAK), or steamer duck decreases the integrin mobile fraction. For rhea and FAK, there is a concomitant shift to Brownian (i.e., time-independent) diffusion at reduced concentrations of these proteins. In contrast, when the expression of actin 42A, dreadlocks, paxillin, integrin-linked kinase (ILK), or vinculin is reduced, integrin diffusion generally becomes more constrained with an increase in the integrin mobile fraction. This same change in integrin diffusion is measured in the absence of integrin extracellular ligand. The results indicate breaking the extracellular ligand-integrin-cytoskeletal linkage alters integrin diffusion properties, and, in most cases, there is no correlation between integrin and lipid diffusion properties.
PTP1B Inhibition Causes Rac1 Activation by Enhancing Receptor Tyrosine Kinase Signaling
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Ayako Tsuchiya
2014-04-01
Full Text Available Background/Aims: The present study investigated the signaling pathway underlying Rac1 activation induced by the linoleic acid derivative 8-[2-(2-pentyl-cyclopropylmethyl-cyclopropyl]-octanoic acid (DCP-LA. Methods: Activity of protein tyrosine phosphatase 1B (PTP1B was assayed under cell-free conditions. Western blot was carried out to quantify phosphorylation of insulin receptor substrate-1 (IRS-1 and Akt in PC-12 cells. Rac1 activity was monitored in the föerster resonance energy transfer (FRET analysis using living and fixed PC-12 cells. Results: DCP-LA markedly suppressed PTP1B activity in a concentration (100 pM-100 µM-dependent manner. In the DCP-LA binding assay, fluorescein-conjugated DCP-LA produced a single fluorescent signal band at 60 kDa, corresponding to the molecule of PTP1B, and the signal was attenuated or abolished by co-treatment or pretreatment with non-conjugated DCP-LA. DCP-LA significantly enhanced nerve growth factor (NGF-stimulated phosphorylation of IRS-1 at Tyr1222 and Akt1/2 at Thr308/309 and Ser473/474 in PC-12 cells. In the FRET analysis, DCP-LA significantly enhanced NGF-stimulated Rac1 activation, which is abrogated by the phosphatidylinositol 3 kinase (PI3K inhibitor wortmannin, the 3-phosphoinositide-dependent protein kinase-1 (PDK1 inhibitor BX912, or the Akt inhibitor MK2206. Conclusion: The results of the present study show that DCP-LA-induced PTP1B inhibition, possibly through its direct binding, causes Rac1 activation by enhancing a pathway along a receptor tyrosine kinase (RTK/IRS-1/PI3K/Akt/Rac1 axis.
Smolders, Sophie Marie-Thérèse; Swinnen, Nina; Kessels, Sofie; Arnauts, Kaline; Smolders, Silke; Le Bras, Barbara; Rigo, Jean-Michel; Legendre, Pascal; Brône, Bert
2017-07-01
Microglia, the immune cells of the central nervous system, take part in brain development and homeostasis. They derive from primitive myeloid progenitors that originate in the yolk sac and colonize the brain mainly through intensive migration. During development, microglial migration speed declines which suggests that their interaction with the microenvironment changes. However, the matrix-cell interactions allowing dispersion within the parenchyma are unknown. Therefore, we aimed to better characterize the migration behavior and to assess the role of matrix-integrin interactions during microglial migration in the embryonic brain ex vivo. We focused on microglia-fibronectin interactions mediated through the fibronectin receptor α5β1 integrin because in vitro work indirectly suggested a role for this ligand-receptor pair. Using 2-photon time-lapse microscopy on acute ex vivo embryonic brain slices, we found that migration occurs in a saltatory pattern and is developmentally regulated. Most importantly, there is an age-specific function of the α5β1 integrin during microglial cortex colonization. At embryonic day (E) 13.5, α5β1 facilitates migration while from E15.5, it inhibits migration. These results indicate a developmentally regulated function of α5β1 integrin in microglial migration during colonization of the embryonic brain. © 2017 Wiley Periodicals, Inc.
Thoh, Maikho; Babajan, Banaganapalli; Raghavendra, Pongali B; Sureshkumar, Chitta; Manna, Sunil K
2011-02-11
Considering the role of retinoids in regulation of more than 500 genes involved in cell cycle and growth arrest, a detailed understanding of the mechanism and its regulation is useful for therapy. The extract of the medicinal plant Neem (Azadirachta indica) is used against several ailments especially for anti-inflammatory, anti-itching, spermicidal, anticancer, and insecticidal activities. In this report we prove the detailed mechanism on the regulation of retinoic acid-mediated cell signaling by azadirachtin, active components of neem extract. Azadirachtin repressed all trans-retinoic acid (ATRA)-mediated nuclear transcription factor κB (NF-κB) activation, not the DNA binding but the NF-κB-dependent gene expression. It did not inhibit IκBα degradation, IκBα kinase activity, or p65 phosphorylation and its nuclear translocation but inhibited NF-κB-dependent reporter gene expression. Azadirachtin inhibited TRAF6-mediated, but not TRAF2-mediated NF-κB activation. It inhibited ATRA-induced Sp1 and CREB (cAMP-response element-binding protein) DNA binding. Azadirachtin inhibited ATRA binding with retinoid receptors, which is supported by biochemical and in silico evidences. Azadirachtin showed strong interaction with retinoid receptors. It suppressed ATRA-mediated removal of retinoid receptors, bound with DNA by inhibiting ATRA binding to its receptors. Overall, our data suggest that azadirachtin interacts with retinoic acid receptors and suppresses ATRA binding, inhibits falling off the receptors, and activates transcription factors like CREB, Sp1, NF-κB, etc. Thus, azadirachtin exerts anti-inflammatory and anti-metastatic responses by a novel pathway that would be beneficial for further anti-inflammatory and anti-cancer therapies.
Thoh, Maikho; Babajan, Banaganapalli; Raghavendra, Pongali B.; Sureshkumar, Chitta; Manna, Sunil K.
2011-01-01
Considering the role of retinoids in regulation of more than 500 genes involved in cell cycle and growth arrest, a detailed understanding of the mechanism and its regulation is useful for therapy. The extract of the medicinal plant Neem (Azadirachta indica) is used against several ailments especially for anti-inflammatory, anti-itching, spermicidal, anticancer, and insecticidal activities. In this report we prove the detailed mechanism on the regulation of retinoic acid-mediated cell signaling by azadirachtin, active components of neem extract. Azadirachtin repressed all trans-retinoic acid (ATRA)-mediated nuclear transcription factor κB (NF-κB) activation, not the DNA binding but the NF-κB-dependent gene expression. It did not inhibit IκBα degradation, IκBα kinase activity, or p65 phosphorylation and its nuclear translocation but inhibited NF-κB-dependent reporter gene expression. Azadirachtin inhibited TRAF6-mediated, but not TRAF2-mediated NF-κB activation. It inhibited ATRA-induced Sp1 and CREB (cAMP-response element-binding protein) DNA binding. Azadirachtin inhibited ATRA binding with retinoid receptors, which is supported by biochemical and in silico evidences. Azadirachtin showed strong interaction with retinoid receptors. It suppressed ATRA-mediated removal of retinoid receptors, bound with DNA by inhibiting ATRA binding to its receptors. Overall, our data suggest that azadirachtin interacts with retinoic acid receptors and suppresses ATRA binding, inhibits falling off the receptors, and activates transcription factors like CREB, Sp1, NF-κB, etc. Thus, azadirachtin exerts anti-inflammatory and anti-metastatic responses by a novel pathway that would be beneficial for further anti-inflammatory and anti-cancer therapies. PMID:21127062
International Nuclear Information System (INIS)
Wilson, K.M.; Minneman, K.P.
1990-01-01
The types of inositol phosphates (InsPs) formed in response to activation of alpha 1-adrenergic receptor subtypes were determined in collagenase-dispersed renal cells and hepatocytes by high pressure liquid chromatography separation. In hepatocytes, which contain only the alpha 1b subtype, norepinephrine stimulated rapid (10-s) formation of [3H]Ins(1,4,5)P3 and [3H]Ins(1,3,4)P3 and slower (5-min) formation of Ins(1,4)P2 and Ins(1)P. Selective inactivation of alpha 1b receptors by chloroethylclonidine almost completely blocked the effects of norepinephrine in hepatocytes. In renal cells, which contain both alpha 1a and alpha 1b receptors in a 60:40 ratio, norepinephrine did not significantly increase the size of any peaks until 5 min after agonist activation. At this time, only a peak eluting with Ins(1)P and one eluting shortly after Ins(1,4)P2 were significantly elevated. Incubation with norepinephrine for 2 h caused small but significant increases in peaks co-eluting with Ins(1)P and Ins(1,4,5)P3 in renal cells; however, only the increase in Ins(1)P was inhibited by chloroethylclonidine pretreatment. Extraction under neutral conditions suggested that cyclic InsPs may be the primary compounds formed in response to norepinephrine in renal cells. Removal of extracellular Ca2+ caused a 60% reduction in the InsP response to norepinephrine in renal cells but had no effect in hepatocytes. These results suggest that activation of alpha 1a and alpha 1b receptor subtypes results in formation of different InsPs and that the response to alpha 1a activation may require influx of extracellular Ca2+
Thys, Mirjan; Nauwynck, Hans; Maes, Dominiek; Hoogewijs, Maarten; Vercauteren, Dries; Rijsselaere, Tom; Favoreel, Herman; Van Soom, Ann
2009-09-01
Fibronectin (Fn) is a 440 kDa glycoprotein assumed to participate in sperm-egg interaction in human. Recently, it has been demonstrated that Fn--when present during bovine IVF--strongly inhibits sperm penetration. The present study was conducted firstly to evaluate the expression of Fn and its integrin receptor (alpha(5)beta(1)) on male and female bovine gametes using indirect immunofluorescence and secondly, to determine the function of Fn during bovine IVF. Endogenous Fn was detected underneath the zona pellucida (ZP) and integrin alpha(5) on the oolemma of cumulus-denuded oocytes. Bovine spermatozoa displayed integrin alpha(5) at their equatorial segment after acrosome reaction. We established that the main inhibitory effect of exogenously supplemented Fn was located at the sperm-oolemma binding, with a (concurrent) effect on fusion, and this can probably be attributed to the binding of Fn to spermatozoa at the equatorial segment, as shown by means of Alexa Fluor 488-conjugated Fn. Combining these results, the inhibitory effect of exogenously supplemented Fn seemed to be exerted on the male gamete by binding to the exposed integrin alpha(5)beta(1) receptor after acrosome reaction. The presence of endogenous Fn underneath the ZP together with integrin alpha(5) expression on oolemma and acrosome-reacted (AR) sperm cell surface suggests a 'velcro' interaction between the endogenous Fn ligand and corresponding receptors on both (AR) sperm cell and oolemma, initiating sperm-egg binding.
DEFF Research Database (Denmark)
Høye, Anette M; Couchman, John R; Wewer, Ulla M
2016-01-01
Integrins, a family of heterodimeric adhesion receptors are implicated in cell migration, development and cancer progression. They can adopt conformations that reflect their activation states and thereby impact adhesion strength and migration. Integrins in an intermediate activation state may be ...
Qin, Xian-Yun; Zhang, Yun-Long; Chi, Ya-Fei; Yan, Bo; Zeng, Xiang-Jun; Li, Hui-Hua; Liu, Ying
2018-01-01
Naive CD4+ T cells differentiate into T helper cells (Th1 and Th2) that play an essential role in the cardiovascular diseases. However, the molecular mechanism by which angiotensin II (Ang II) promotes Th1 differentiation remains unclear. The aim of this study was to determine whether the Ang II-induced Th1 differentiation regulated by ubiquitin-proteasome system (UPS). Jurkat cells were treated with Ang II (100 nM) in the presence or absence of different inhibitors. The gene mRNA levels were detected by real-time quantitative PCR analysis. The protein levels were measured by ELISA assay or Western blot analysis, respectively. Ang II treatment significantly induced a shift from Th0 to Th1 cell differentiation, which was markedly blocked by angiotensin II type 1 receptor (AT1R) inhibitor Losartan (LST). Moreover, Ang II significantly increased the activities and the expression of proteasome catalytic subunits (β1, β1i, β2i and β5i) in a dose- and time-dependent manner. However, Ang II-induced proteasome activities were remarkably abrogated by LST and PKA inhibitor H-89. Mechanistically, Ang II-induced Th1 differentiation was at least in part through proteasome-mediated degradation of IκBα and MKP-1 and activation of STAT1 and NF-κB. This study for the first time demonstrates that Ang II activates AT1R-PKA-proteasome pathway, which promotes degradation of IκBα and MKP-1 and activation of STAT1 and NF-κB thereby leading to Th1 differentiation. Thus, inhibition of proteasome activation might be a potential therapeutic target for Th1-mediated diseases. © 2018 The Author(s). Published by S. Karger AG, Basel.
Integrin activation dynamics between the RGD-binding site and the headpiece hinge.
Puklin-Faucher, Eileen; Vogel, Viola
2009-12-25
Integrins form mechanical links between the extracellular matrix and the cytoskeleton. Although integrin activation is known to be regulated by an allosteric conformational change, which can be induced from the extracellular or intracellular end of the molecule, little is known regarding the sequence of structural events by which signals propagate between distant sites. Here, we reveal with molecular dynamics simulations of the FnIII(10)-bound alpha(V)beta(3) integrin headpiece how the binding pocket and interdomain betaA/hybrid domain hinge on the distal end of the betaA domain are allosterically linked via a hydrophobic T-junction between the middle of the alpha1 helix and top of the alpha7 helix. The key results of this study are: 1) that this T-junction is induced by ligand binding and hinge opening, and thus displays bidirectionality; 2) that formation of this junction can be accelerated by ligand-mediated force; and 3) how formation of this junction is inhibited by Ca(2+) in place of Mg(2+) at the site adjacent to the metal ion-dependent adhesion site ("ADMIDAS"). Together with recent experimental evidence that integrin complexes can form catch bonds (i.e. become strengthened under force), as well as earlier evidence that Ca(2+) at the ADMIDAS results in lower binding affinity, these simulations provide a common structural model for the dynamic process by which integrins become activated.
Aono, Yuri; Kiguchi, Yuri; Watanabe, Yuriko; Waddington, John L; Saigusa, Tadashi
2017-11-15
The nucleus accumbens contains delta-opioid receptors that may reduce inhibitory neurotransmission. Reduction in GABA A receptor-mediated inhibition of accumbal dopamine release due to delta-opioid receptor activation should be suppressed by stimulating accumbal GABA A receptors. As delta-opioid receptors are divided into delta2- and delta1-opioid receptors, we analysed the effects of the GABA A receptor agonist muscimol on delta2- and delta1-opioid receptor-mediated accumbal dopamine efflux in freely moving rats using in vivo microdialysis. Drugs were administered intracerebrally through the dialysis probe. Doses of compounds indicate total amount administered (mol) during 25-50min infusions. The delta2-opioid receptor agonist deltorphin II (25.0nmol)- and delta1-opioid receptor agonist DPDPE (5.0nmol)-induced increases in dopamine efflux were inhibited by the delta2-opioid receptor antagonist naltriben (1.5nmol) and the delta1-opioid receptor antagonist BNTX (150.0pmol), respectively. Muscimol (250.0pmol) inhibited deltorphin II (25.0nmol)-induced dopamine efflux. The GABA A receptor antagonist bicuculline (50.0pmol), which failed to affect deltorphin II (25.0nmol)-induced dopamine efflux, counteracted the inhibitory effect of muscimol on deltorphin II-induced dopamine efflux. Neither muscimol (250.0pmol) nor bicuculline (50.0 and 500.0pmol) altered DPDPE (5.0nmol)-induced dopamine efflux. The present results show that reduction in accumbal GABA A receptor-mediated inhibition of dopaminergic activity is necessary to produce delta2-opioid receptor-induced increase in accumbal dopamine efflux. This study indicates that activation of delta2- but not delta1-opioid receptors on the cell bodies and/or terminals of accumbal GABAergic interneurons inhibits GABA release and, accordingly, decreases GABA A receptor-mediated inhibition of dopaminergic terminals, resulting in enhanced accumbal dopamine efflux. Copyright © 2017 Elsevier B.V. All rights reserved.
Directory of Open Access Journals (Sweden)
César Martín
Full Text Available Bordetella pertussis, the bacterium that causes whooping cough, secretes an adenylate cyclase toxin (ACT that must be post-translationally palmitoylated in the bacterium cytosol to be active. The toxin targets phagocytes expressing the CD11b/CD18 integrin receptor. It delivers a catalytic adenylate cyclase domain into the target cell cytosol producing a rapid increase of intracellular cAMP concentration that suppresses bactericidal functions of the phagocyte. ACT also induces calcium fluxes into target cells. Biochemical, biophysical and cell biology approaches have been applied here to show evidence that ACT and integrin molecules, along with other raft components, are rapidly internalized by the macrophages in a toxin-induced calcium rise-dependent process. The toxin-triggered internalisation events occur through two different routes of entry, chlorpromazine-sensitive receptor-mediated endocytosis and clathrin-independent internalisation, maybe acting in parallel. ACT locates into raft-like domains, and is internalised, also in cells devoid of receptor. Altogether our results suggest that adenylate cyclase toxin, and maybe other homologous pathogenic toxins from the RTX (Repeats in Toxin family to which ACT belongs, may be endowed with an intrinsic capacity to, directly and efficiently, insert into raft-like domains, promoting there its multiple activities. One direct consequence of the integrin removal from the cell surface of the macrophages is the hampering of their adhesion ability, a fundamental property in the immune response of the leukocytes that could be instrumental in the pathogenesis of Bordetella pertussis.
Martín, César; Uribe, Kepa B; Gómez-Bilbao, Geraxane; Ostolaza, Helena
2011-02-23
Bordetella pertussis, the bacterium that causes whooping cough, secretes an adenylate cyclase toxin (ACT) that must be post-translationally palmitoylated in the bacterium cytosol to be active. The toxin targets phagocytes expressing the CD11b/CD18 integrin receptor. It delivers a catalytic adenylate cyclase domain into the target cell cytosol producing a rapid increase of intracellular cAMP concentration that suppresses bactericidal functions of the phagocyte. ACT also induces calcium fluxes into target cells. Biochemical, biophysical and cell biology approaches have been applied here to show evidence that ACT and integrin molecules, along with other raft components, are rapidly internalized by the macrophages in a toxin-induced calcium rise-dependent process. The toxin-triggered internalisation events occur through two different routes of entry, chlorpromazine-sensitive receptor-mediated endocytosis and clathrin-independent internalisation, maybe acting in parallel. ACT locates into raft-like domains, and is internalised, also in cells devoid of receptor. Altogether our results suggest that adenylate cyclase toxin, and maybe other homologous pathogenic toxins from the RTX (Repeats in Toxin) family to which ACT belongs, may be endowed with an intrinsic capacity to, directly and efficiently, insert into raft-like domains, promoting there its multiple activities. One direct consequence of the integrin removal from the cell surface of the macrophages is the hampering of their adhesion ability, a fundamental property in the immune response of the leukocytes that could be instrumental in the pathogenesis of Bordetella pertussis.
Foubert, Philippe; Silvestre, Jean-Sébastien; Souttou, Boussad; Barateau, Véronique; Martin, Coralie; Ebrahimian, Téni G.; Leré-Déan, Carole; Contreres, Jean Olivier; Sulpice, Eric; Levy, Bernard I.; Plouët, Jean; Tobelem, Gérard; Le Ricousse-Roussanne, Sophie
2007-01-01
Endothelial progenitor cell (EPC) transplantation has beneficial effects for therapeutic neovascularization; however, only a small proportion of injected cells home to the lesion and incorporate into the neocapillaries. Consequently, this type of cell therapy requires substantial improvement to be of clinical value. Erythropoietin-producing human hepatocellular carcinoma (Eph) receptors and their ephrin ligands are key regulators of vascular development. We postulated that activation of the EphB4/ephrin-B2 system may enhance EPC proangiogenic potential. In this report, we demonstrate in a nude mouse model of hind limb ischemia that EphB4 activation with an ephrin-B2–Fc chimeric protein increases the angiogenic potential of human EPCs. This effect was abolished by EphB4 siRNA, confirming that it is mediated by EphB4. EphB4 activation enhanced P selectin glycoprotein ligand-1 (PSGL-1) expression and EPC adhesion. Inhibition of PSGL-1 by siRNA reversed the proangiogenic and adhesive effects of EphB4 activation. Moreover, neutralizing antibodies to E selectin and P selectin blocked ephrin-B2–Fc–stimulated EPC adhesion properties. Thus, activation of EphB4 enhances EPC proangiogenic capacity through induction of PSGL-1 expression and adhesion to E selectin and P selectin. Therefore, activation of EphB4 is an innovative and potentially valuable therapeutic strategy for improving the recruitment of EPCs to sites of neovascularization and thereby the efficiency of cell-based proangiogenic therapy. PMID:17510705
Sangboonruang, S; Thammasit, P; Intasai, N; Kasinrerk, W; Tayapiwatana, C; Tragoolpua, K
2014-06-01
Extracellular matrix metalloproteinase inducer (EMMPRIN) exhibits overexpression in various cancers and promotes cancer progression and metastasis via the interaction with its associated molecules. The scFv-M6-1B9 intrabody has a potential ability to reduce EMMPRIN cell surface expression. However, the subsequent effect of scFv-M6-1B9 intrabody-mediated EMMPRIN abatement on its related molecules, α3β1-integrin, MCT1, MMP-2 and MMP-9, is undefined. Our results demonstrated that the scFv-M6-1B9 intrabody efficiently decreased α3β1-integrin cell surface expression levels. In addition, intracellular accumulation of MCT1 and lactate were increased. These results lead to suppression of features characteristic for tumor progression, including cell migration, proliferation and invasion, in a colorectal cancer cell line (Caco-2) although there was no difference in MMP expression. Thus, EMMPRIN represents an attractive target molecule for the disruption of cancer proliferation and metastasis. An scFv-M6-1B9 intrabody-based approach could be relevant for cancer gene therapy.
Barker, Thomas H; Baneyx, Gretchen; Cardó-Vila, Marina; Workman, Gail A; Weaver, Matt; Menon, Priya M; Dedhar, Shoukat; Rempel, Sandra A; Arap, Wadih; Pasqualini, Renata; Vogel, Viola; Sage, E Helene
2005-10-28
SPARC, a 32-kDa matricellular glycoprotein, mediates interactions between cells and their extracellular matrix, and targeted deletion of Sparc results in compromised extracellular matrix in mice. Fibronectin matrix provides provisional tissue scaffolding during development and wound healing and is essential for the stabilization of mature extracellular matrix. Herein, we report that SPARC expression does not significantly affect fibronectin-induced cell spreading but enhances fibronectin-induced stress fiber formation and cell-mediated partial unfolding of fibronectin molecules, an essential process in fibronectin matrix assembly. By phage display, we identify integrin-linked kinase as a potential binding partner of SPARC and verify the interaction by co-immunoprecipitation and colocalization in vitro. Cells lacking SPARC exhibit diminished fibronectin-induced integrin-linked kinase activation and integrin-linked kinase-dependent cell-contractile signaling. Furthermore, induced expression of SPARC in SPARC-null fibroblasts restores fibronectin-induced integrin-linked kinase activation, downstream signaling, and fibronectin unfolding. These data further confirm the function of SPARC in extracellular matrix organization and identify a novel mechanism by which SPARC regulates extracellular matrix assembly.
IMPDHII Protein Inhibits Toll-like Receptor 2-mediated Activation of NF-κB*
Toubiana, Julie; Rossi, Anne-Lise; Grimaldi, David; Belaidouni, Nadia; Chafey, Philippe; Clary, Guilhem; Courtine, Emilie; Pene, Frederic; Mira, Jean-Paul; Claessens, Yann-Erick; Chiche, Jean-Daniel
2011-01-01
Toll-like receptor 2 (TLR2) plays an essential role in innate immunity by the recognition of a large variety of pathogen-associated molecular patterns. It induces its recruitment to lipid rafts induces the formation of a membranous activation cluster necessary to enhance, amplify, and control downstream signaling. However, the exact composition of the TLR2-mediated molecular complex is unknown. We performed a proteomic analysis in lipopeptide-stimulated THP1 and found IMPDHII protein rapidly recruited to lipid raft. Whereas IMPDHII is essential for lymphocyte proliferation, its biologic function within innate immune signal pathways has not been established yet. We report here that IMPDHII plays an important role in the negative regulation of TLR2 signaling by modulating PI3K activity. Indeed, IMPDHII increases the phosphatase activity of SHP1, which participates to the inactivation of PI3K. PMID:21460227
Liver X receptor α and farnesoid X receptor are major transcriptional regulators of OATP1B1.
Meyer Zu Schwabedissen, Henriette E; Böttcher, Kerstin; Chaudhry, Amarjit; Kroemer, Heyo K; Schuetz, Erin G; Kim, Richard B
2010-11-01
Organic anion transporting polypeptide 1B1 (OATP1B1) is a liver-enriched transporter involved in the hepatocellular uptake of many endogenous molecules and several structurally divergent drugs in clinical use. Although OATP1B1 coding region polymorphisms are known to make an impact on substrate drug disposition in humans, little is known regarding the mechanisms underlying the transcriptional regulation of this transporter. In this study, we note that messenger RNA (mRNA) expression of OATP1B1 in a large human liver bank exhibited marked interindividual variability that was not associated with coding region polymorphisms. Accordingly, we hypothesized that such variability in expression is reflective of nuclear receptor-mediated transcriptional regulation of this transporter. We tested prototypical ligands for the nuclear receptors pregnane X receptor (PXR), constitutive androstane receptor (CAR), liver X receptor (LXR) α, and farnesoid X receptor (FXR) in a human hepatoma-derived cell line and noted induction of OATP1B1 mRNA when the cells were treated with LXRα or FXR ligands. To confirm a direct role for LXRα and FXR to OATP1B1 expression, we performed detailed promoter analysis and cell-based reporter gene assays resulting in the identification of two functional FXR response elements and one LXRα response element. The direct interaction between nuclear receptors with the identified response elements was assessed using chromatin immunoprecipitation assays. Using isolated primary human hepatocytes, we show that LXRα or FXR agonists, but not PXR or CAR agonists, are capable of OATP1B1 induction. We note that OATP1B1 transcriptional regulation is under dual nuclear receptor control through the oxysterol sensing LXRα and the bile acid sensor FXR. Accordingly, the interplay between OATP1B1 and nuclear receptors may play an important and heretofore unrecognized role during cholestasis, drug-induced liver injury, and OATP1B1 induction-related drug interactions.
Blumbach, Katrin; Niehoff, Anja; Belgardt, Bengt F.; Ehlen, Harald W. A.; Schmitz, Markus; Hallinger, Ralf; Schulz, Jan-Niklas; Brüning, Jens C.; Krieg, Thomas; Schubert, Markus; Gullberg, Donald; Eckes, Beate
2012-01-01
Mice with a combined deficiency in the α2β1 and α11β1 integrins lack the major receptors for collagen I. These mutants are born with inconspicuous differences in size but develop dwarfism within the first 4 weeks of life. Dwarfism correlates with shorter, less mineralized and functionally weaker bones that do not result from growth plate abnormalities or osteoblast dysfunction. Besides skeletal dwarfism, internal organs are correspondingly smaller, indicating proportional dwarfism and suggesting a systemic cause for the overall size reduction. In accordance with a critical role of insulin-like growth factor (IGF)-1 in growth control and bone mineralization, circulating IGF-1 levels in the sera of mice lacking either α2β1 or α11β1 or both integrins were sharply reduced by 39%, 64%, or 81% of normal levels, respectively. Low hepatic IGF-1 production resulted from diminished growth hormone-releasing hormone expression in the hypothalamus and, subsequently, reduced growth hormone expression in the pituitary glands of these mice. These findings point out a novel role of collagen-binding integrin receptors in the control of growth hormone/IGF-1-dependent biological activities. Thus, coupling hormone secretion to extracellular matrix signaling via integrins represents a novel concept in the control of endocrine homeostasis. PMID:22210772
Blumbach, Katrin; Niehoff, Anja; Belgardt, Bengt F; Ehlen, Harald W A; Schmitz, Markus; Hallinger, Ralf; Schulz, Jan-Niklas; Brüning, Jens C; Krieg, Thomas; Schubert, Markus; Gullberg, Donald; Eckes, Beate
2012-02-24
Mice with a combined deficiency in the α2β1 and α11β1 integrins lack the major receptors for collagen I. These mutants are born with inconspicuous differences in size but develop dwarfism within the first 4 weeks of life. Dwarfism correlates with shorter, less mineralized and functionally weaker bones that do not result from growth plate abnormalities or osteoblast dysfunction. Besides skeletal dwarfism, internal organs are correspondingly smaller, indicating proportional dwarfism and suggesting a systemic cause for the overall size reduction. In accordance with a critical role of insulin-like growth factor (IGF)-1 in growth control and bone mineralization, circulating IGF-1 levels in the sera of mice lacking either α2β1 or α11β1 or both integrins were sharply reduced by 39%, 64%, or 81% of normal levels, respectively. Low hepatic IGF-1 production resulted from diminished growth hormone-releasing hormone expression in the hypothalamus and, subsequently, reduced growth hormone expression in the pituitary glands of these mice. These findings point out a novel role of collagen-binding integrin receptors in the control of growth hormone/IGF-1-dependent biological activities. Thus, coupling hormone secretion to extracellular matrix signaling via integrins represents a novel concept in the control of endocrine homeostasis.
Directory of Open Access Journals (Sweden)
Dan L
2012-10-01
Full Text Available Li Dan,1,* Ding Jian,2,* Lin Na,1 Wang Xiaozhong,1 1Digestive Department, the Union Hospital of Fujian Medical University, Fujian, People’s Republic of China; 2Digestive Department, the First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian, People’s Republic of China*These authors contributed equally to this workBackground/objective: To investigate the crosstalk between epidermal growth factor receptor (EGFR and integrin-mediated signal transduction pathways in human gastric adenocarcinoma cells.Methods: EGF was used as a ligand of EGFR to stimulate the gastric adenocarcinoma cell, SGC7901. Signal molecules downstream of the integrin, FAK(Y397 and p130cas(Y410 phosphorylation, were measured by immunoprecipitation and western blot. Fibronectin (Fn was used as a ligand of integrin to stimulate the same cell line. Signal molecules downstream of EGFR and extracellular signal-regulated kinase (ERK general phosphorylation were also measured. Focal adhesion kinase (FAK small-interfering RNA was designed and transfected into SGC7901 cells to decrease the expression of FAK. Modified Boyden chambers and MTT assay were used to examine the effect of FAK inhibition on the invasiveness and proliferation of SGC7901.Results: EGF activated FAK(Y397 and p130cas(Y410 phosphorylation, while Fn activated ERK general phosphorylation. Inhibition of FAK expression decreased p130cas(Y410 phosphorylation activated by EGF and ERK general phosphorylation activated by Fn, also decreased the invasiveness and proliferation of SGC7901 cells activated by EGF or Fn.Conclusion: There is crosstalk between EGFR and integrin signal transduction. FAK may be a key cross point of the two signal pathways and acts as a potential target for human gastric cancer therapy.Keywords: gastric adenocarcinoma, epidermal growth factor receptor, integrin, focal adhesion kinase, crosstalk
Energy Technology Data Exchange (ETDEWEB)
Do, Minh Truong; Kim, Hyung Gyun; Tran, Thi Thu Phuong; Khanal, Tilak; Choi, Jae Ho [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of); Chung, Young Chul [Department of Food Science and Culinary, International University of Korea, Jinju (Korea, Republic of); Jeong, Tae Cheon, E-mail: taecheon@ynu.ac.kr [College of Pharmacy, Yeungnam University, Gyeongsan (Korea, Republic of); Jeong, Hye Gwang, E-mail: hgjeong@cnu.ac.kr [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of)
2014-10-01
Induction of cytochrome P450 (CYP) 1A1 and CYP1B1 by environmental xenobiotic chemicals or endogenous ligands through the activation of the aryl hydrocarbon receptor (AhR) has been implicated in a variety of cellular processes related to cancer, such as transformation and tumorigenesis. Here, we investigated the effects of the anti-diabetes drug metformin on expression of CYP1A1 and CYP1B1 in breast cancer cells under constitutive and inducible conditions. Our results indicated that metformin down-regulated the expression of CYP1A1 and CYP1B1 in breast cancer cells under constitutive and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced conditions. Down-regulation of AhR expression was required for metformin-mediated decreases in CYP1A1 and CYP1B1 expression, and the metformin-mediated CYP1A1 and CYP1B1 reduction is irrelevant to estrogen receptor α (ERα) signaling. Furthermore, we found that metformin markedly down-regulated Sp1 protein levels in breast cancer cells. The use of genetic and pharmacological tools revealed that metformin-mediated down-regulation of AhR expression was mediated through the reduction of Sp1 protein. Metformin inhibited endogenous AhR ligand-induced CYP1A1 and CYP1B1 expression by suppressing tryptophan-2,3-dioxygenase (TDO) expression in MCF-7 cells. Finally, metformin inhibits TDO expression through a down-regulation of Sp1 and glucocorticoid receptor (GR) protein levels. Our findings demonstrate that metformin reduces CYP1A1 and CYP1B1 expression in breast cancer cells by down-regulating AhR signaling. Metformin would be able to act as a potential chemopreventive agent against CYP1A1 and CYP1B1-mediated carcinogenesis and development of cancer. - Graphical abstract: Schematic of the CYP1A1 and CYP1B1 gene regulation by metformin. - Highlights: • Metformin inhibits CYP1A1 and CYP1B1 expression. • Metformin down-regulates the AhR signaling. • Metformin reduces Sp1 protein expression. • Metformin suppresses TDO expression.
International Nuclear Information System (INIS)
Do, Minh Truong; Kim, Hyung Gyun; Tran, Thi Thu Phuong; Khanal, Tilak; Choi, Jae Ho; Chung, Young Chul; Jeong, Tae Cheon; Jeong, Hye Gwang
2014-01-01
Induction of cytochrome P450 (CYP) 1A1 and CYP1B1 by environmental xenobiotic chemicals or endogenous ligands through the activation of the aryl hydrocarbon receptor (AhR) has been implicated in a variety of cellular processes related to cancer, such as transformation and tumorigenesis. Here, we investigated the effects of the anti-diabetes drug metformin on expression of CYP1A1 and CYP1B1 in breast cancer cells under constitutive and inducible conditions. Our results indicated that metformin down-regulated the expression of CYP1A1 and CYP1B1 in breast cancer cells under constitutive and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced conditions. Down-regulation of AhR expression was required for metformin-mediated decreases in CYP1A1 and CYP1B1 expression, and the metformin-mediated CYP1A1 and CYP1B1 reduction is irrelevant to estrogen receptor α (ERα) signaling. Furthermore, we found that metformin markedly down-regulated Sp1 protein levels in breast cancer cells. The use of genetic and pharmacological tools revealed that metformin-mediated down-regulation of AhR expression was mediated through the reduction of Sp1 protein. Metformin inhibited endogenous AhR ligand-induced CYP1A1 and CYP1B1 expression by suppressing tryptophan-2,3-dioxygenase (TDO) expression in MCF-7 cells. Finally, metformin inhibits TDO expression through a down-regulation of Sp1 and glucocorticoid receptor (GR) protein levels. Our findings demonstrate that metformin reduces CYP1A1 and CYP1B1 expression in breast cancer cells by down-regulating AhR signaling. Metformin would be able to act as a potential chemopreventive agent against CYP1A1 and CYP1B1-mediated carcinogenesis and development of cancer. - Graphical abstract: Schematic of the CYP1A1 and CYP1B1 gene regulation by metformin. - Highlights: • Metformin inhibits CYP1A1 and CYP1B1 expression. • Metformin down-regulates the AhR signaling. • Metformin reduces Sp1 protein expression. • Metformin suppresses TDO expression
International Nuclear Information System (INIS)
Wang, Yanling; Fälting, Johanna M.; Mattsson, Charlotte L.; Holmström, Therése E.; Nedergaard, Jan
2013-01-01
Brown adipose tissue is unusual in that the neurotransmitter norepinephrine influences cell destiny in ways generally associated with effects of classical growth factors: regulation of cell proliferation, of apoptosis, and progression of differentiation. The norepinephrine effects are mediated through G-protein-coupled receptors; further mediation of such stimulation to e.g. Erk1/2 activation is in cell biology in general accepted to occur through transactivation of the EGF receptor (by external or internal pathways). We have examined here the significance of such transactivation in brown adipocytes. Stimulation of mature brown adipocytes with cirazoline (α 1 -adrenoceptor coupled via G q ), clonidine (α 2 via G i ) or CL316243 (β 3 via G s ) or via β 1 -receptors significantly activated Erk1/2. Pretreatment with the EGF receptor kinase inhibitor AG1478 had, remarkably, no significant effect on Erk1/2 activation induced by any of these adrenergic agonists (although it fully abolished EGF-induced Erk1/2 activation), demonstrating absence of EGF receptor-mediated transactivation. Results with brown preadipocytes (cells in more proliferative states) were not qualitatively different. Joint stimulation of all adrenoceptors with norepinephrine did not result in synergism on Erk1/2 activation. AG1478 action on EGF-stimulated Erk1/2 phosphorylation showed a sharp concentration–response relationship (IC 50 0.3 µM); a minor apparent effect of AG1478 on norepinephrine-stimulated Erk1/2 phosphorylation showed nonspecific kinetics, implying caution in interpretation of partial effects of AG1478 as reported in other systems. Transactivation of the EGF receptor is clearly not a universal prerequisite for coupling of G-protein coupled receptors to Erk1/2 signalling cascades. - Highlights: • In brown adipocytes, norepinephrine regulates proliferation, apoptosis, differentiation. • EGF receptor transactivation is supposed to mediate GPCR-induced Erk1/2 activation. •
Directory of Open Access Journals (Sweden)
Mohammad Harun-Or-Rashid
Full Text Available Injury to the eye or retina triggers Müller cells, the major glia cell of the retina, to dedifferentiate and proliferate. In some species they attain retinal progenitor properties and have the capacity to generate new neurons. The epidermal growth factor receptor (EGFR system and extracellular signal-regulated kinase (ERK signaling are key regulators of these processes in Müller cells. The extracellular signals that modulate and control these processes are not fully understood. In this work we studied whether endothelin receptor signaling can activate EGFR and ERK signaling in Müller cells. Endothelin expression is robustly upregulated at retinal injury and endothelin receptors have been shown to transactivate EGFRs in other cell types. We analyzed the endothelin signaling system in chicken retina and cultured primary chicken Müller cells as well as the human Müller cell line MIO-M1. The Müller cells were stimulated with receptor agonists and treated with specific blockers to key enzymes in the signaling pathway or with siRNAs. We focused on endothelin receptor mediated transactivation of EGFRs by using western blot analysis, quantitative reverse transcriptase PCR and immunocytochemistry. The results showed that chicken Müller cells and the human Müller cell line MIO-M1 express endothelin receptor B. Stimulation by the endothelin receptor B agonist IRL1620 triggered phosphorylation of ERK1/2 and autophosphorylation of (Y1173 EGFR. The effects could be blocked by Src-kinase inhibitors (PP1, PP2, EGFR-inhibitor (AG1478, EGFR-siRNA and by inhibitors to extracellular matrix metalloproteinases (GM6001, consistent with a Src-kinase mediated endothelin receptor response that engage ligand-dependent and ligand-independent EGFR activation. Our data suggest a mechanism for how injury-induced endothelins, produced in the retina, may modulate the Müller cell responses by Src-mediated transactivation of EGFRs. The data give support to a view in
Recruitment of activation receptors at inhibitory NK cell immune synapses.
Directory of Open Access Journals (Sweden)
Nicolas Schleinitz
2008-09-01
Full Text Available Natural killer (NK cell activation receptors accumulate by an actin-dependent process at cytotoxic immune synapses where they provide synergistic signals that trigger NK cell effector functions. In contrast, NK cell inhibitory receptors, including members of the MHC class I-specific killer cell Ig-like receptor (KIR family, accumulate at inhibitory immune synapses, block actin dynamics, and prevent actin-dependent phosphorylation of activation receptors. Therefore, one would predict inhibition of actin-dependent accumulation of activation receptors when inhibitory receptors are engaged. By confocal imaging of primary human NK cells in contact with target cells expressing physiological ligands of NK cell receptors, we show here that this prediction is incorrect. Target cells included a human cell line and transfected Drosophila insect cells that expressed ligands of NK cell activation receptors in combination with an MHC class I ligand of inhibitory KIR. The two NK cell activation receptors CD2 and 2B4 accumulated and co-localized with KIR at inhibitory immune synapses. In fact, KIR promoted CD2 and 2B4 clustering, as CD2 and 2B4 accumulated more efficiently at inhibitory synapses. In contrast, accumulation of KIR and of activation receptors at inhibitory synapses correlated with reduced density of the integrin LFA-1. These results imply that inhibitory KIR does not prevent CD2 and 2B4 signaling by blocking their accumulation at NK cell immune synapses, but by blocking their ability to signal within inhibitory synapses.
Differential expression of integrins and laminin-5 in normal oral epithelia
DEFF Research Database (Denmark)
Thorup, A K; Dabelsteen, Erik; Schou, S
1997-01-01
beta 1 and beta 4 integrins are receptors on epithelial cells mediating cell-extracellular matrix adhesion. Furthermore, alpha 2 beta 1 and alpha 3 beta 1 contribute to cell-cell adhesion. Laminin-5 in epithelial basement membranes (BMs) is a ligand for alpha 6 beta 4 and alpha 3 beta 1. Expressi...
Fujita, Masaaki; Ieguchi, Katsuaki; Davari, Parastoo; Yamaji, Satoshi; Taniguchi, Yukimasa; Sekiguchi, Kiyotoshi; Takada, Yoko K.; Takada, Yoshikazu
2012-01-01
Background: Integrin αvβ3-extracellular matrix interaction and/or αvβ3 binding to insulin-like growth factor-1 (IGF1; and integrin-IGF1-IGF1 receptor ternary complex formation) is critical for IGF signaling.
Bidirectional remodeling of β1-integrin adhesions during chemotropic regulation of nerve growth
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Carlstrom Lucas P
2011-11-01
Full Text Available Abstract Background Chemotropic factors in the extracellular microenvironment guide nerve growth by acting on the growth cone located at the tip of extending axons. Growth cone extension requires the coordination of cytoskeleton-dependent membrane protrusion and dynamic adhesion to the extracellular matrix, yet how chemotropic factors regulate these events remains an outstanding question. We demonstrated previously that the inhibitory factor myelin-associated glycoprotein (MAG triggers endocytic removal of the adhesion receptor β1-integrin from the growth cone surface membrane to negatively remodel substrate adhesions during chemorepulsion. Here, we tested how a neurotrophin might affect integrin adhesions. Results We report that brain-derived neurotropic factor (BDNF positively regulates the formation of substrate adhesions in axonal growth cones during stimulated outgrowth and prevents removal of β1-integrin adhesions by MAG. Treatment of Xenopus spinal neurons with BDNF rapidly triggered β1-integrin clustering and induced the dynamic formation of nascent vinculin-containing adhesion complexes in the growth cone periphery. Both the formation of nascent β1-integrin adhesions and the stimulation of axon extension by BDNF required cytoplasmic calcium ion signaling and integrin activation at the cell surface. Exposure to MAG decreased the number of β1-integrin adhesions in the growth cone during inhibition of axon extension. In contrast, the BDNF-induced adhesions were resistant to negative remodeling by MAG, correlating with the ability of BDNF pretreatment to counteract MAG-inhibition of axon extension. Pre-exposure to MAG prevented the BDNF-induced formation of β1-integrin adhesions and blocked the stimulation of axon extension by BDNF. Conclusions Altogether, these findings demonstrate the neurotrophin-dependent formation of integrin-based adhesions in the growth cone and reveal how a positive regulator of substrate adhesions can block
Coalition of Oct4A and β1 integrins in facilitating metastasis in ovarian cancer
International Nuclear Information System (INIS)
Samardzija, Chantel; Luwor, Rodney B.; Quinn, Michael A.; Kannourakis, George; Findlay, Jock K.; Ahmed, Nuzhat
2016-01-01
Ovarian cancer is a metastatic disease and one of the leading causes of gynaecology malignancy-related deaths in women. Cancer stem cells (CSCs) are key contributors of cancer metastasis and relapse. Integrins are a family of cell surface receptors which allow interactions between cells and their surrounding microenvironment and play a fundamental role in promoting metastasis. This study investigates the molecular mechanism which associates CSCs and integrins in ovarian cancer metastasis. The expression of Oct4A in high-grade serous ovarian tumors and normal ovaries was determined by immunofluorescence analysis. The functional role of Oct4A was evaluated by generating stable knockdown (KD) of Oct4A clones in an established ovarian cancer cell line HEY using shRNA-mediated silencing. The expression of integrins in cell lines was evaluated by flow cytometry. Spheroid forming ability, adhesion and the activities of matrix metalloproteinases 9/2 (MMP-9/2) was measured by in vitro functional assays and gelatin zymography. These observations were further validated in in vivo mouse models using Balb/c nu/nu mice. We report significantly elevated expression of Oct4A in high-grade serous ovarian tumors compared to normal ovarian tissues. The expression of Oct4A in ovarian cancer cell lines correlated with their CSC-related sphere forming abilities. The suppression of Oct4A in HEY cells resulted in a significant diminution of integrin β1 expression and associated α5 and α2 subunits compared to vector control cells. This was associated with a reduced adhesive ability on collagen and fibronectin and decreased secretion of pro-MMP2 in Oct4A KD cells compared to vector control cells. In vivo, Oct4A knock down (KD) cells produced tumors which were significantly smaller in size and weight compared to tumors derived from vector control cells. Immunohistochemical analyses of Oct4A KD tumor xenografts demonstrated a significant loss of cytokeratin 7 (CK7), Glut-1 as well as CD34
Integrin Activation Dynamics between the RGD-binding Site and the Headpiece Hinge*
Puklin-Faucher, Eileen; Vogel, Viola
2009-01-01
Integrins form mechanical links between the extracellular matrix and the cytoskeleton. Although integrin activation is known to be regulated by an allosteric conformational change, which can be induced from the extracellular or intracellular end of the molecule, little is known regarding the sequence of structural events by which signals propagate between distant sites. Here, we reveal with molecular dynamics simulations of the FnIII10-bound αVβ3 integrin headpiece how the binding pocket and interdomain βA/hybrid domain hinge on the distal end of the βA domain are allosterically linked via a hydrophobic T-junction between the middle of the α1 helix and top of the α7 helix. The key results of this study are: 1) that this T-junction is induced by ligand binding and hinge opening, and thus displays bidirectionality; 2) that formation of this junction can be accelerated by ligand-mediated force; and 3) how formation of this junction is inhibited by Ca2+ in place of Mg2+ at the site adjacent to the metal ion-dependent adhesion site (“ADMIDAS”). Together with recent experimental evidence that integrin complexes can form catch bonds (i.e. become strengthened under force), as well as earlier evidence that Ca2+ at the ADMIDAS results in lower binding affinity, these simulations provide a common structural model for the dynamic process by which integrins become activated. PMID:19762919
Dickreuter, E.; Eke, I.; Krause, M.; Borgmann, K.; van Vugt, M. A.; Cordes, N.
2016-01-01
beta 1 Integrin-mediated cell-extracellular matrix interactions allow cancer cell survival and confer therapy resistance. It was shown that inhibition of beta 1 integrins sensitizes cells to radiotherapy. Here, we examined the impact of beta 1 integrin targeting on the repair of radiation-induced
Endocytosis of Integrin-Binding Human Picornaviruses
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Pirjo Merilahti
2012-01-01
Full Text Available Picornaviruses that infect humans form one of the largest virus groups with almost three hundred virus types. They include significant enteroviral pathogens such as rhino-, polio-, echo-, and coxsackieviruses and human parechoviruses that cause wide range of disease symptoms. Despite the economic importance of picornaviruses, there are no antivirals. More than ten cellular receptors are known to participate in picornavirus infection, but experimental evidence of their role in cellular infection has been shown for only about twenty picornavirus types. Three enterovirus types and one parechovirus have experimentally been shown to bind and use integrin receptors in cellular infection. These include coxsackievirus A9 (CV-A9, echovirus 9, and human parechovirus 1 that are among the most common and epidemic human picornaviruses and bind to αV-integrins via RGD motif that resides on virus capsid. In contrast, echovirus 1 (E-1 has no RGD and uses integrin α2β1 as cellular receptor. Endocytosis of CV-A9 has recently been shown to occur via a novel Arf6- and dynamin-dependent pathways, while, contrary to collagen binding, E-1 binds inactive β1 integrin and enters via macropinocytosis. In this paper, we review what is known about receptors and endocytosis of integrin-binding human picornaviruses.
Impaired P2X1 Receptor-Mediated Adhesion in Eosinophils from Asthmatic Patients.
Wright, Adam; Mahaut-Smith, Martyn; Symon, Fiona; Sylvius, Nicolas; Ran, Shaun; Bafadhel, Mona; Muessel, Michelle; Bradding, Peter; Wardlaw, Andrew; Vial, Catherine
2016-06-15
Eosinophils play an important role in the pathogenesis of asthma and can be activated by extracellular nucleotides released following cell damage or inflammation. For example, increased ATP concentrations were reported in bronchoalveolar lavage fluids of asthmatic patients. Although eosinophils are known to express several subtypes of P2 receptors for extracellular nucleotides, their function and contribution to asthma remain unclear. In this article, we show that transcripts for P2X1, P2X4, and P2X5 receptors were expressed in healthy and asthmatic eosinophils. The P2X receptor agonist α,β-methylene ATP (α,β-meATP; 10 μM) evoked rapidly activating and desensitizing inward currents (peak 18 ± 3 pA/pF at -60 mV) in healthy eosinophils, typical of P2X1 homomeric receptors, which were abolished by the selective P2X1 antagonist NF449 (1 μM) (3 ± 2 pA/pF). α,β-meATP-evoked currents were smaller in eosinophils from asthmatic patients (8 ± 2 versus 27 ± 5 pA/pF for healthy) but were enhanced following treatment with a high concentration of the nucleotidase apyrase (17 ± 5 pA/pF for 10 IU/ml and 11 ± 3 pA/pF for 0.32 IU/ml), indicating that the channels are partially desensitized by extracellular nucleotides. α,β-meATP (10 μM) increased the expression of CD11b activated form in eosinophils from healthy, but not asthmatic, donors (143 ± 21% and 108 ± 11% of control response, respectively). Furthermore, α,β-meATP increased healthy (18 ± 2% compared with control 10 ± 1%) but not asthmatic (13 ± 1% versus 10 ± 0% for control) eosinophil adhesion. Healthy human eosinophils express functional P2X1 receptors whose activation leads to eosinophil αMβ2 integrin-dependent adhesion. P2X1 responses are constitutively reduced in asthmatic compared with healthy eosinophils, probably as the result of an increase in extracellular nucleotide concentration. Copyright © 2016 by The American Association of Immunologists, Inc.
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Jakubowski, Piotr; Calvete, Juan J.; Eble, Johannes A.; Lazarovici, Philip; Marcinkiewicz, Cezary
2013-01-01
Snake venom antagonists of α2β1 integrin have been identified as members of a C-lectin type family of proteins (CLP). In the present study, we characterized three new CLPs isolated from Echis sochureki venom, which interact with this integrin. These proteins were purified using a combination of gel filtration, ion exchange chromatography and reverse phase HPLC. Sochicetin-A and sochicetin-B potently inhibited adhesion of cells expressing α2β1 integrin and binding of isolated α2β1 ectodomain to collagen I, as well as bound to recombinant GST-α2A domain in ELISA, whereas activity of sochicetin-C in these assays was approximately two orders of magnitude lower. Structurally, sochicetin-B and sochicetin-C are typical heterodimeric αβ CLPs, whereas sochicetin-A exhibits a trimer of its subunits (αβ) 3 in the quaternary structure. Immobilized sochicetins supported adhesion of glioma cell lines, LN18 and LBC3, whereas in a soluble form they partially inhibited adhesion of these cells to collagen I. Glioma cells spread very poorly on sochicetin-A, showing no cytoskeleton rearrangement typical for adhesion to collagen I or fibronectin. Adhesion on CLP does not involve focal adhesion elements, such as vinculin. Sochicetin-A also inhibited collagen-induced platelet aggregation, similar to other CLPs' action on the blood coagulation system. - Highlights: • Isolation of three novel snake venom CLPs inhibiting α2β1 integrin • Reporting hexameric CLP, sochicetin-A with anti-collagen receptor activity • CLPs antagonize the interaction of glioma cells with collagen matrix. • Sochicetin-A does not support glioma cell spreading
Energy Technology Data Exchange (ETDEWEB)
Jakubowski, Piotr [Temple University, Department of Biology, Philadelphia, PA 19122 (United States); Calvete, Juan J. [Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Cientificas, 46010 Valencia (Spain); Eble, Johannes A. [Excellence Cluster Cardio-Pulmonary System, Center for Molecular Medicine, Vascular Matrix Biology, Frankfurt University Hospital, Frankfurt am Main 60590 (Germany); Lazarovici, Philip [The Hebrew University of Jerusalem, School of Pharmacy, Institute for Drug Research, Jerusalem 91120 (Israel); Marcinkiewicz, Cezary, E-mail: cmarcink@temple.edu [Temple University, Department of Biology, Philadelphia, PA 19122 (United States)
2013-05-15
Snake venom antagonists of α2β1 integrin have been identified as members of a C-lectin type family of proteins (CLP). In the present study, we characterized three new CLPs isolated from Echis sochureki venom, which interact with this integrin. These proteins were purified using a combination of gel filtration, ion exchange chromatography and reverse phase HPLC. Sochicetin-A and sochicetin-B potently inhibited adhesion of cells expressing α2β1 integrin and binding of isolated α2β1 ectodomain to collagen I, as well as bound to recombinant GST-α2A domain in ELISA, whereas activity of sochicetin-C in these assays was approximately two orders of magnitude lower. Structurally, sochicetin-B and sochicetin-C are typical heterodimeric αβ CLPs, whereas sochicetin-A exhibits a trimer of its subunits (αβ){sub 3} in the quaternary structure. Immobilized sochicetins supported adhesion of glioma cell lines, LN18 and LBC3, whereas in a soluble form they partially inhibited adhesion of these cells to collagen I. Glioma cells spread very poorly on sochicetin-A, showing no cytoskeleton rearrangement typical for adhesion to collagen I or fibronectin. Adhesion on CLP does not involve focal adhesion elements, such as vinculin. Sochicetin-A also inhibited collagen-induced platelet aggregation, similar to other CLPs' action on the blood coagulation system. - Highlights: • Isolation of three novel snake venom CLPs inhibiting α2β1 integrin • Reporting hexameric CLP, sochicetin-A with anti-collagen receptor activity • CLPs antagonize the interaction of glioma cells with collagen matrix. • Sochicetin-A does not support glioma cell spreading.
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Nicoletta Zoppi
2018-03-01
Full Text Available The αvβ3 integrin, an endothelial cells’ receptor-binding fibronectin (FN in the extracellular matrix (ECM of blood vessels, regulates ECM remodeling during migration, invasion, angiogenesis, wound healing and inflammation, and is also involved in the epithelial mesenchymal transition. In vitro-grown human control fibroblasts organize a fibrillar network of FN, which is preferentially bound on the entire cell surface to its canonical α5β1 integrin receptor, whereas the αvβ3 integrin is present only in rare patches in focal contacts. We report on the preferential recruitment of the αvβ3 integrin, due to the lack of FN–ECM and its canonical integrin receptor, in dermal fibroblasts from Ehlers–Danlos syndromes (EDS and arterial tortuosity syndrome (ATS, which are rare multisystem connective tissue disorders. We review our previous findings that unraveled different biological mechanisms elicited by the αvβ3 integrin in fibroblasts derived from patients affected with classical (cEDS, vascular (vEDS, hypermobile EDS (hEDS, hypermobility spectrum disorders (HSD, and ATS. In cEDS and vEDS, respectively, due to defective type V and type III collagens, αvβ3 rescues patients’ fibroblasts from anoikis through a paxillin-p60Src-mediated cross-talk with the EGF receptor. In hEDS and HSD, without a defined molecular basis, the αvβ3 integrin transduces to the ILK-Snail1-axis inducing a fibroblast-to-myofibroblast-transition. In ATS cells, the deficiency of the dehydroascorbic acid transporter GLUT10 leads to redox imbalance, ECM disarray together with the activation of a non-canonical αvβ3 integrin-TGFBRII signaling, involving p125FAK/p60Src/p38MAPK. The characterization of these different biological functions triggered by αvβ3 provides insights into the multifaced nature of this integrin, at least in cultured dermal fibroblasts, offering future perspectives for research in this field.
Activation of TRPV1 by capsaicin induces functional Kinin B1 receptor in rat spinal cord microglia
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Talbot Sébastien
2012-01-01
Full Text Available Abstract Background The kinin B1 receptor (B1R is upregulated by pro-inflammatory cytokines and oxydative stress, which are enhanced by transient receptor potential vanilloid subtype 1 (TRPV1 activation. To examine the link between TRPV1 and B1R in inflammatory pain, this study aimed to determine the ability of TRPV1 to regulate microglial B1R expression in the spinal cord dorsal horn, and the underlying mechanism. Methods B1R expression (mRNA, protein and binding sites was measured in cervical, thoracic and lumbar spinal cord in response to TRPV1 activation by systemic capsaicin (1-50 mg/kg, s.c in rats pre-treated with TRPV1 antagonists (capsazepine or SB-366791, the antioxidant N-acetyl-L-cysteine (NAC, or vehicle. B1R function was assessed using a tail-flick test after intrathecal (i.t. injection of a selective B1R agonist (des-Arg9-BK, and its microglial localization was investigated by confocal microscopy with the selective fluorescent B1R agonist, [Nα-bodipy]-des-Arg9-BK. The effect of i.t. capsaicin (1 μg/site was also investigated. Results Capsaicin (10 to 50 mg/kg, s.c. enhanced time-dependently (0-24h B1R mRNA levels in the lumbar spinal cord; this effect was prevented by capsazepine (10 mg/kg, i.p.; 10 μg/site, i.t. and SB-366791 (1 mg/kg, i.p.; 30 μg/site, i.t.. Increases of B1R mRNA were correlated with IL-1β mRNA levels, and they were significantly less in cervical and thoracic spinal cord. Intrathecal capsaicin (1 μg/site also enhanced B1R mRNA in lumbar spinal cord. NAC (1 g/kg/d × 7 days prevented B1R up-regulation, superoxide anion production and NF-kB activation induced by capsaicin (15 mg/kg. Des-Arg9-BK (9.6 nmol/site, i.t. decreased by 25-30% the nociceptive threshold at 1 min post-injection in capsaicin-treated rats (10-50 mg/kg while it was without effect in control rats. Des-Arg9-BK-induced thermal hyperalgesia was blocked by capsazepine, SB-366791 and by antagonists/inhibitors of B1R (SSR240612, 10 mg/kg, p
Yadav, S K; Haldar, C; Singh, S S
2011-12-01
Daily variation in the peripheral level of melatonin plays a major role in integrating reproduction and environmental information for seasonally breeding birds. However, the variation in immunity and reproduction has never been assessed in any avian species on a 24 h time scale. Therefore, to understand the relationship between immune function and reproductive phases in a seasonally breeding bird, Perdicula asiatica, the Indian jungle bush quail, we studied the daily variation of melatonin and testosterone levels along with expression of their receptors Mel(1a), Mel(1b), and androgen receptor in the spleen during the reproductively active phase. Immunocytochemistry for the melatonin receptors Mel(1a) and Mel(1b) presented a differential distribution pattern. Western blot of splenic protein suggested a daily rhythm of melatonin receptors, while acrophases for the two melatonin receptors Mel(1a) and Mel(1b) differed by 4 h, suggesting that the expression of the receptors may peak at different times, causing more of either Mel(1a) or Mel(1b) to be available at a particular time to mediate function. The circulatory melatonin level correlated with percentage stimulation ratio of splenocytes and plasma interleukin-2 level, but did not correlate with testosterone or androgen receptor, suggesting that melatonin could be a major hormone imparting a time-of-day effect on the modulation of immune function in a seasonally breeding bird during the reproductively active phase. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.
B-Raf and CRHR1 internalization mediate biphasic ERK1/2 activation by CRH in hippocampal HT22 Cells.
Bonfiglio, Juan J; Inda, Carolina; Senin, Sergio; Maccarrone, Giuseppina; Refojo, Damián; Giacomini, Damiana; Turck, Christoph W; Holsboer, Florian; Arzt, Eduardo; Silberstein, Susana
2013-03-01
CRH is a key regulator of neuroendocrine, autonomic, and behavioral response to stress. CRH-stimulated CRH receptor 1 (CRHR1) activates ERK1/2 depending on intracellular context. In a previous work, we demonstrated that CRH activates ERK1/2 in limbic areas of the mouse brain (hippocampus and basolateral amygdala). ERK1/2 is an essential mediator of hippocampal physiological processes including emotional behavior, synaptic plasticity, learning, and memory. To elucidate the molecular mechanisms by which CRH activates ERK1/2 in hippocampal neurons, we used the mouse hippocampal cell line HT22. We document for the first time that ERK1/2 activation in response to CRH is biphasic, involving a first cAMP- and B-Raf-dependent early phase and a second phase that critically depends on CRHR1 internalization and β-arrestin2. By means of mass-spectrometry-based screening, we identified B-Raf-associated proteins that coimmunoprecipitate with endogenous B-Raf after CRHR1 activation. Using molecular and pharmacological tools, the functional impact of selected B-Raf partners in CRH-dependent ERK1/2 activation was dissected. These results indicate that 14-3-3 proteins, protein kinase A, and Rap1, are essential for early CRH-induced ERK1/2 activation, whereas dynamin and vimentin are required for the CRHR1 internalization-dependent phase. Both phases of ERK1/2 activation depend on calcium influx and are affected by calcium/calmodulin-dependent protein kinase II inactivation. Thus, this report describes the dynamics and biphasic nature of ERK1/2 activation downstream neuronal CRHR1 and identifies several new critical components of the CRHR1 signaling machinery that selectively controls the early and late phases of ERK1/2 activation, thus providing new potential therapeutic targets for stress-related disorders.
Beta1 integrins regulate chondrocyte rotation, G1 progression, and cytokinesis
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Aszodi, Attila; Hunziker, Ernst B; Brakebusch, Cord
2003-01-01
Beta1 integrins are highly expressed on chondrocytes, where they mediate adhesion to cartilage matrix proteins. To assess the functions of beta1 integrin during skeletogenesis, we inactivated the beta1 integrin gene in chondrocytes. We show here that these mutant mice develop a chondrodysplasia...... of various severity. beta1-deficient chondrocytes had an abnormal shape and failed to arrange into columns in the growth plate. This is caused by a lack of motility, which is in turn caused by a loss of adhesion to collagen type II, reduced binding to and impaired spreading on fibronectin, and an abnormal F......-actin organization. In addition, mutant chondrocytes show decreased proliferation caused by a defect in G1/S transition and cytokinesis. The G1/S defect is, at least partially, caused by overexpression of Fgfr3, nuclear translocation of Stat1/Stat5a, and up-regulation of the cell cycle inhibitors p16 and p21...
Allain, Fabrice; Vanpouille, Christophe; Carpentier, Mathieu; Slomianny, Marie-Christine; Durieux, Sandrine; Spik, Geneviève
2002-01-01
Cyclophilins A and B (CyPA and CyPB) are cyclosporin A-binding proteins that are involved in inflammatory events. We have reported that CyPB interacts with two types of cell-surface-binding sites. The first site corresponds to a functional receptor and requires interaction with the central core of CyPB. This region is highly conserved in cyclophilins, suggesting that CyPA and CyPB might share biological activities mediated by interaction with this receptor. The second site is identified with ...
Genetic analysis of beta1 integrin "activation motifs" in mice
DEFF Research Database (Denmark)
Czuchra, Aleksandra; Meyer, Hannelore; Legate, Kyle R
2006-01-01
-null phenotype in vivo. Surprisingly, neither the substitution of the tyrosines with phenylalanine nor the aspartic acid with alanine resulted in an obvious defect. These data suggest that the NPXY motifs of the beta1 integrin tail are essential for beta1 integrin function, whereas tyrosine phosphorylation...
Case, Lindsay B.; Waterman, Clare M.
2011-01-01
At the leading lamellipodium of migrating cells, protrusion of an Arp2/3-nucleated actin network is coupled to formation of integrin-based adhesions, suggesting that Arp2/3-mediated actin polymerization and integrin-dependent adhesion may be mechanistically linked. Arp2/3 also mediates actin polymerization in structures distinct from the lamellipodium, in "ventral F-actin waves" that propagate as spots and wavefronts along the ventral plasma membrane. Here we show that integrins engage the ex...
Niu, Gengming; Ye, Taiyang; Qin, Liuliang; Bourbon, Pierre M; Chang, Cheng; Zhao, Shengqiang; Li, Yan; Zhou, Lei; Cui, Pengfei; Rabinovitz, Issac; Mercurio, Arthur M; Zhao, Dezheng; Zeng, Huiyan
2015-01-01
Tissue repair/wound healing, in which angiogenesis plays an important role, is a critical step in many diseases including chronic wound, myocardial infarction, stroke, cancer, and inflammation. Recently, we were the first to report that orphan nuclear receptor TR3/Nur77 is a critical mediator of angiogenesis and its associated microvessel permeability. Tumor growth and angiogenesis induced by VEGF-A, histamine, and serotonin are almost completely inhibited in Nur77 knockout mice. However, it is not known whether TR3/Nur77 plays any roles in wound healing. In these studies, skin wound-healing assay was performed in 3 types of genetically modified mice having various Nur77 activities. We found that ectopic induction of Nur77 in endothelial cells of mice is sufficient to improve skin wound healing. Although skin wound healing in Nur77 knockout mice is comparable to the wild-type control mice, the process is significantly delayed in the EC-Nur77-DN mice, in which a dominant negative Nur77 mutant is inducibly and specifically expressed in mouse endothelial cells. By a loss-of-function assay, we elucidate a novel feed-forward signaling pathway, integrin β4 → PI3K → Akt → FAK, by which TR3 mediates HUVEC migration. Furthermore, TR3/Nur77 regulates the expression of integrin β4 by targeting its promoter activity. In conclusion, expression of TR3/Nur77 improves wound healing by targeting integrin β4. TR3/Nur77 is a potential candidate for proangiogenic therapy. The results further suggest that TR3/Nur77 is required for pathologic angiogenesis but not for developmental/physiologic angiogenesis and that Nur77 and its family members play a redundant role in normal skin wound healing. © FASEB.
Knop, J; Wesche, H; Lang, D; Martin, M U
1998-10-01
The association and activation of the IL-1 receptor-associated protein kinase (IRAK) to the IL-1 receptor complex is one of the earliest events detectable in IL-1 signal transduction. We generated permanent clones of the murine T cell line EL4 6.1 overexpressing human (h)IRAK to evaluate the role of this kinase in IL-1 signaling. Overexpression of hIRAK enhanced IL-1-stimulated activation of the transcription factor NFkappaB, whereas a truncated form (N-IRAK) specifically inhibited IL-1-dependent NFkappaB activity. In clones stably overexpressing hIRAK a weak constitutive activation of NFkappaB correlated with a low basal IL-2 production which was enhanced in an IL-1-dependent manner. Compared to the parental cell line the dose-response curve of IL-1-induced IL-2 production was shifted in both potency and efficacy. These results demonstrate that IRAK directly triggers NFkappaB-mediated gene expression in EL4 cells. Qualitatively different effects were observed for the IL-1-induced activation of stress-activated protein (SAP) kinases: permanent overexpression of IRAK did not affect the dose dependence but prolonged the kinetics of IL-1-induced activation of SAP kinases, suggesting that this signaling branch may be regulated by distinct mechanisms.
Miyazaki, Tatsuya; Miyauchi, Satoshi; Anada, Takahisa; Tawada, Akira; Suzuki, Osamu
2015-10-01
Integrins and their ligands have been suggested to be associated with osteoclast-mediated bone resorption. The present study was designed to investigate whether chondroitin sulfate E (CS-E), which is one of the sulfated glycosaminoglycans (GAGs), is involved in osteoactivin (OA) activity, and osteoclast differentiation. The binding affinity of sulfated GAGs to integrin and its ligand was measured using biotin-labeled CS-E, and the osteoclast differentiation was evaluated by tartrate-resistant acid phosphatase staining and a pit formation assay. CS-E as well as CS-B, synthetic chondroitin polysulfate, and heparin inhibited osteoclast differentiation of bone marrow-derived macrophages. Pre-coating of OA to synthetic calcium phosphate-coated plates enhanced the osteoclastic differentiation of RAW264 cells, and addition of a neutralizing antibody to OA inhibited its differentiation. CS-E bound not only to OA, fibronectin, and vitronectin, but also to its receptor integrin αVβ3, and inhibited the direct binding of OA to integrin αVβ3. Furthermore, CS-E blocked the binding of OA to cells and inhibited OA-induced osteoclastic differentiation. On the other hand, heparinase treatment of RAW264 cells inhibited osteoclastic differentiation. Since binding of OA to the cells was inhibited by the presence of heparan sulfate or heparinase treatment of cells, heparan sulfate proteoglycan (HSPG) was also considered to be an OA receptor. Taken together, the present results suggest that CS-E is capable of inhibiting OA-induced osteoclast differentiation by blocking the interaction of OA to integrin αVβ3 and HSPG. © 2015 Wiley Periodicals, Inc.
DEFF Research Database (Denmark)
Zhang, Wei; Zhang, Yaping; Edvinsson, Lars
2009-01-01
culture of the arteries, VSMC TP receptors were studied by using myography, real-time PCR and immunohistochemistry. We observed that organ culture for 24 and 48 h resulted in depressed TP receptor-mediated contraction in the VSMC, in parallel with decreased TP receptor mRNA and protein expressions....... Phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2), p38 and nuclear factor-kappaB (NF-kappaB) was seen by Western blot within 1-3 h after organ culture. Inhibition of ERK1/2, p38 or NF-kappaB reversed depressed contraction as well as decreased receptor mRNA expression. Actinomycin D...
DEFF Research Database (Denmark)
Zhang, Wei; Zhang, Yaping; Edvinsson, Lars
2008-01-01
culture of the arteries, VSMC TP receptors were studied by using myography, real-time PCR and immunohistochemistry. We observed that organ culture for 24 and 48 h resulted in depressed TP receptor-mediated contraction in the VSMC, in parallel with decreased TP receptor mRNA and protein expressions....... Phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2), p38 and nuclear factor-kappaB (NF-kappaB) was seen by Western blot within 1-3 h after organ culture. Inhibition of ERK1/2, p38 or NF-kappaB reversed depressed contraction as well as decreased receptor mRNA expression. Actinomycin D...
Johansson, Mats W.; Gunderson, Kristin A.; Kelly, Elizabeth A. B.; Denlinger, Loren C.; Jarjour, Nizar N.; Mosher, Deane F.
2013-01-01
Background IL-5 activates αMβ2 integrin on blood eosinophils in vitro. Eosinophils in bronchoalveolar lavage (BAL) following segmental antigen challenge have activated β2-integrins. Objective To identify roles for IL-5 in regulating human eosinophil integrins in vivo. Methods Blood and BAL eosinophils were analyzed by flow cytometry in ten subjects with allergic asthma who underwent a segmental antigen challenge protocol before and after anti-IL-5 administration. Results Blood eosinophil reactivity with monoclonal antibody (mAb) KIM-127, which recognizes partially activated β2-integrins, was decreased after anti-IL-5. Before anti-IL-5, surface densities of blood eosinophil β2, αM, and αL integrin subunits increased modestly post-challenge. After anti-IL-5, such increases did not occur. Before or after anti-IL-5, surface densities of β2,αM, αL, and αD and reactivity with KIM-127 and mAb CBRM1/5, which recognizes high-activity αMβ2, were similarly high on BAL eosinophils 48 h post-challenge. Density and activation state of β1-integrins on blood and BAL eosinophils were not impacted by anti-IL-5, even though anti-IL-5 ablated a modest post-challenge increase on blood or BAL eosinophils of P-selectin glycoprotein ligand-1 (PSGL-1), a receptor for P-selectin that causes activation of β1-integrins. Forward scatter of blood eosinophils post-challenge was less heterogeneous and on the average decreased after anti-IL-5; however, anti-IL-5 had no effect on the decreased forward scatter of eosinophils in post-challenge BAL compared to eosinophils in blood. Blood eosinophil KIM-127 reactivity at the time of challenge correlated with the percentage of eosinophils in BAL post-challenge. Conclusion and Clinical Relevance IL-5 supports a heterogeneous population of circulating eosinophils with partially activated β2-integrins and is responsible for upregulation of β2-integrins and PSGL-1 on circulating eosinophils following segmental antigen challenge but has
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Marcinkiewicz C
2017-05-01
Full Text Available Cezary Marcinkiewicz,1,2 Jonathan A Gerstenhaber,1 Mark Sternberg,2 Peter I Lelkes,1 Giora Feuerstein1,2 1Department of Bioengineering, College of Engineering, Temple University, Philadelphia, 2Debina Diagnostic, Inc., Newton Square, PA, USA Abstract: Thromboembolic events (TEE underwrite key causes of death in developed countries. While advanced imaging technologies such as computed tomography scans serve to diagnose blood clots during acute cardiovascular events, no such technology is available in routine primary care for TEE risk assessment. Here, we describe an imaging platform technology based on bioengineered fluorescent nanodiamond particles (F-NDPs functionalized with bitistatin (Bit, a disintegrin that specifically binds to the αIIbβ3 integrin, platelet fibrinogen receptor (PFR on activated platelets. Covalent linkage of purified Bit to F-NDP was concentration-dependent and saturable, as validated by enzyme-linked immunosorbent assay using specific anti-Bit antibodies. F-NDP–Bit interacted with purified PFR, either in immobilized or soluble form. Lotrafiban, a nonpeptide, αIIbβ3 receptor antagonist, specifically blocked F-NDP–Bit–PFR complex formation. Moreover, F-NDP–Bit specifically binds to activated platelets incorporated into a clot generated by thrombin-activated rat platelet-rich plasma (PRP. Our results suggest that engineered F-NDP–Bit particles could serve as noninvasive, “real-time” optical diagnostics for clots present in blood vessels. Keywords: carbon nanoparticles, blood clots, imaging, platelet fibrinogen receptor, fluorescence, disintegrin, thromboembolic complications, thrombosis
The Rho-family GTPase Rac1 regulates integrin localization in Drosophila immunosurveillance cells.
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Miguel J Xavier
Full Text Available BACKGROUND: When the parasitoid wasp Leptopilina boulardi lays an egg in a Drosophila larva, phagocytic cells called plasmatocytes and specialized cells known as lamellocytes encapsulate the egg. The Drosophila β-integrin Myospheroid (Mys is necessary for lamellocytes to adhere to the cellular capsule surrounding L. boulardi eggs. Integrins are heterodimeric adhesion receptors consisting of α and β subunits, and similar to other plasma membrane receptors undergo ligand-dependent endocytosis. In mammalian cells it is known that integrin binding to the extracellular matrix induces the activation of Rac GTPases, and we have previously shown that Rac1 and Rac2 are necessary for a proper encapsulation response in Drosophila larvae. We wanted to test the possibility that Myospheroid and Rac GTPases interact during the Drosophila anti-parasitoid immune response. RESULTS: In the current study we demonstrate that Rac1 is required for the proper localization of Myospheroid to the cell periphery of haemocytes after parasitization. Interestingly, the mislocalization of Myospheroid in Rac1 mutants is rescued by hyperthermia, involving the heat shock protein Hsp83. From these results we conclude that Rac1 and Hsp83 are required for the proper localization of Mys after parasitization. SIGNIFICANCE: We show for the first time that the small GTPase Rac1 is required for Mysopheroid localization. Interestingly, the necessity of Rac1 in Mys localization was negated by hyperthermia. This presents a problem, in Drosophila we quite often raise larvae at 29°C when using the GAL4/UAS misexpression system. If hyperthermia rescues receptor endosomal recycling defects, raising larvae in hyperthermic conditions may mask potentially interesting phenotypes.
Jiang, Xue; Yang, Jingwen; Shen, Zhangfei; Chen, Yajie; Shi, Liangen; Zhou, Naiming
2016-08-01
Diapause is a developmental strategy adopted by insects to survive in challenging environments such as the low temperatures of a winter. This unique process is regulated by diapause hormone (DH), which is a neuropeptide hormone that induces egg diapause in Bombyx mori and is involved in terminating pupal diapause in heliothis moths. An G protein-coupled receptor from the silkworm, B. mori, has been identified as a specific cell surface receptor for DH. However, the detailed information on the DH-DHR system and its mechanism(s) involved in the induction of embryonic diapause remains unknown. Here, we combined functional assays with various specific inhibitors to elucidate the DHR-mediated signaling pathways. Upon activation by DH, B. mori DHR is coupled to the Gq protein, leading to a significant increase of intracellular Ca(2+) and cAMP response element-driven luciferase activity in an UBO-QIC, a specific Gq inhibitor, sensitive manner. B. mori DHR elicited ERK1/2 phosphorylation in a dose- and time-dependent manner in response to DH. This effect was almost completely inhibited by co-incubation with UBO-QIC and was also significantly suppressed by PLC inhibitor U73122, PKC inhibitors Gö6983 and the Ca(2+) chelator EGTA. Moreover, DHR-induced activation of ERK1/2 was significantly attenuated by treatment with the Gβγ specific inhibitors gallein and M119K and the PI3K specific inhibitor Wortmannin, but not by the Src specific inhibitor PP2. Our data also demonstrates that the EGFR-transactivation pathway is not involved in the DHR-mediated ERK1/2 phosphorylation. Future efforts are needed to clarify the role of the ERK1/2 signaling pathway in the DH-mediated induction of B. mori embryonic diapause. Copyright © 2016 Elsevier Ltd. All rights reserved.
UNC93B1 mediates host resistance to infection with Toxoplasma gondii.
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Mariane B Melo
2010-08-01
Full Text Available UNC93B1 associates with Toll-Like Receptor (TLR 3, TLR7 and TLR9, mediating their translocation from the endoplasmic reticulum to the endolysosome, hence allowing proper activation by nucleic acid ligands. We found that the triple deficient '3d' mice, which lack functional UNC93B1, are hyper-susceptible to infection with Toxoplasma gondii. We established that while mounting a normal systemic pro-inflammatory response, i.e. producing abundant MCP-1, IL-6, TNFα and IFNγ, the 3d mice were unable to control parasite replication. Nevertheless, infection of reciprocal bone marrow chimeras between wild-type and 3d mice with T. gondii demonstrated a primary role of hemopoietic cell lineages in the enhanced susceptibility of UNC93B1 mutant mice. The protective role mediated by UNC93B1 to T. gondii infection was associated with impaired IL-12 responses and delayed IFNγ by spleen cells. Notably, in macrophages infected with T. gondii, UNC93B1 accumulates on the parasitophorous vacuole. Furthermore, upon in vitro infection the rate of tachyzoite replication was enhanced in non-activated macrophages carrying mutant UNC93B1 as compared to wild type gene. Strikingly, the role of UNC93B1 on intracellular parasite growth appears to be independent of TLR function. Altogether, our results reveal a critical role for UNC93B1 on induction of IL-12/IFNγ production as well as autonomous control of Toxoplasma replication by macrophages.
SNARE-mediated trafficking of α5β1 integrin is required for spreading in CHO cells
International Nuclear Information System (INIS)
Skalski, Michael; Coppolino, Marc G.
2005-01-01
In this study, the role of SNARE-mediated membrane traffic in regulating integrin localization was examined and the requirement for SNARE function in cellular spreading was quantitatively assessed. Membrane traffic was inhibited with the VAMP-specific catalytic light chain from tetanus toxin (TeTx-LC), a dominant-negative form (E329Q) of N-ethylmaleimide-sensitive fusion protein (NSF), and brefeldin A (BfA). Inhibition of membrane traffic with either E329Q-NSF or TeTx-LC, but not BfA, significantly inhibited spreading of CHO cells on fibronectin. Spreading was rescued in TeTx-LC-expressing cells by co-transfection with a TeTx-resistant cellubrevin/VAMP3. E329Q-NSF, a general inhibitor of SNARE function, was a more potent inhibitor of cell spreading than TeTx-LC, suggesting that tetanus toxin-insensitive SNAREs contribute to adhesion. It was found that E329Q-NSF prevented trafficking of α 5 β 1 integrins from a central Rab11-containing compartment to sites of protrusion during cell adhesion, while TeTx-LC delayed this trafficking. These results are consistent with a model of cellular adhesion that implicates SNARE function as an important component of integrin trafficking during the process of cell spreading
Zhang, Yi-Xin; Li, Xiao-Fang; Yuan, Guo-Qiang; Hu, Hui; Song, Xiao-Yun; Li, Jing-Yi; Miao, Xiao-Kang; Zhou, Tian-Xiong; Yang, Wen-Le; Zhang, Xiao-Wei; Mou, Ling-Yun; Wang, Rui
2017-05-26
Glioblastoma is the most common malignant brain tumor and has a poor prognosis. Tachykinin receptor neurokinin-1 (NK1R) is a promising target in glioblastoma therapy because of its overexpression in human glioblastoma. NK1R agonists promote glioblastoma cell growth, whereas NK1R antagonists efficiently inhibit cell growth both in vitro and in vivo However, the molecular mechanisms involved in these effects are incompletely understood. β-Arrestins (ARRBs) serve as scaffold proteins and adapters to mediate intracellular signal transduction. Here we show that the ARRB1-mediated signaling pathway is essential for NK1-mediated glioblastoma cell proliferation. ARRB1 knockdown significantly inhibited NK1-mediated glioblastoma cell proliferation and induced G 2 /M phase cell cycle arrest. ARRB1 knockdown cells showed remarkable down-regulation of CDC25C/CDK1/cyclin B1 activity. We also demonstrated that ARRB1 mediated prolonged phosphorylation of ERK1/2 and Akt in glioblastoma cells induced by NK1R activation. ERK1/2 and Akt phosphorylation are involved in regulating CDC25C/CDK1/cyclin B1 activity. The lack of long-term ERK1/2 and Akt activation in ARRB1 knockdown cells was at least partly responsible for the delayed cell cycle progression and proliferation. Moreover, we found that ARRB1-mediated ERK1/2 and Akt phosphorylation regulated the transcriptional activity of both NF-κB and AP-1, which were involved in cyclin B1 expression. ARRB1 deficiency increased the sensitivity of glioblastoma cells to the treatment of NK1R antagonists. Taken together, our results suggest that ARRB1 plays an essential role in NK1R-mediated cell proliferation and G 2 /M transition in glioblastoma cells. Interference with ARRB1-mediated signaling via NK1R may have potential significance for therapeutic strategies targeting glioblastoma. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
The newcomer in the integrin family: Integrin α9 in biology and cancer
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Høye, Anette Melissa; Couchman, John Robert; Wewer, Ulla M.
2012-01-01
Integrins are heterodimeric transmembrane receptors regulating cell-cell and cell-extracellular matrix interactions. Of the 24 integrin heterodimers identified in humans, a9ß1 integrin is one of the least studied. a9, together with a4, comprise a more recent evolutionary sub-family of integrins...... of cell types, interacts with many ligands for example fibronectin, tenascin-C and ADAM12, and has been shown to have important functions in processes such as cell adhesion and migration, lung development, lymphatic and venous valve development, and in wound healing. This has sparked an interest...
Protease activated receptors (PARS) mediation in gyroxin biological activity
International Nuclear Information System (INIS)
Silva, Jose Alberto Alves da
2009-01-01
Gyroxin is a serine protease enzyme from the South American rattlesnake (Crotalus durissus terrificus) venom; it is only partially characterized and has multiple activities. Gyroxin induces blood coagulation, blood pressure decrease and a neurotoxic behavior named barrel rotation. The mechanisms involved in this neurotoxic activity are not known. Whereas gyroxin is a member of enzymes with high potential to become a new drug with clinical applications such as thrombin, batroxobin, ancrod, tripsyn and kalicrein, it is important to find out how gyroxin works. The analysis on agarose gel electrophoresis and circular dichroism confirmed the molecules' integrity and purity. The gyroxin intravenous administration in mice proved its neurotoxicity (barrel rotation). In vivo studies employing intravital microscopy proved that gyroxin induces vasodilation with the participation of protease activated receptors (PARs), nitric oxide and Na+K+ATPase. The leukocytes' adherence and rolling counting indicated that gyroxin has no pro inflammatory activity. Gyroxin induced platelet aggregation, which was blocked by inhibitors of PAR1 and PAR4 receptors (SCH 79797 and tcY-NH 2 , respectively). Finally, it was proved that the gyroxin temporarily alter the permeability of the blood brain barrier (BBB). Our study has shown that both the protease-activated receptors and nitric oxide are mediators involved in the biological activities of gyroxin. (author)
Kindlins, integrin activation and the regulation of talin recruitment to αIIbβ3.
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Bryan N Kahner
Full Text Available Talins and kindlins bind to the integrin β3 cytoplasmic tail and both are required for effective activation of integrin αIIbβ3 and resulting high-affinity ligand binding in platelets. However, binding of the talin head domain alone to β3 is sufficient to activate purified integrin αIIbβ3 in vitro. Since talin is localized to the cytoplasm of unstimulated platelets, its re-localization to the plasma membrane and to the integrin is required for activation. Here we explored the mechanism whereby kindlins function as integrin co-activators. To test whether kindlins regulate talin recruitment to plasma membranes and to αIIbβ3, full-length talin and kindlin recruitment to β3 was studied using a reconstructed CHO cell model system that recapitulates agonist-induced αIIbβ3 activation. Over-expression of kindlin-2, the endogenous kindlin isoform in CHO cells, promoted PAR1-mediated and talin-dependent ligand binding. In contrast, shRNA knockdown of kindlin-2 inhibited ligand binding. However, depletion of kindlin-2 by shRNA did not affect talin recruitment to the plasma membrane, as assessed by sub-cellular fractionation, and neither over-expression of kindlins nor depletion of kindlin-2 affected talin interaction with αIIbβ3 in living cells, as monitored by bimolecular fluorescence complementation. Furthermore, talin failed to promote kindlin-2 association with αIIbβ3 in CHO cells. In addition, purified talin and kindlin-3, the kindlin isoform expressed in platelets, failed to promote each other's binding to the β3 cytoplasmic tail in vitro. Thus, kindlins do not promote initial talin recruitment to αIIbβ3, suggesting that they co-activate integrin through a mechanism independent of recruitment.
International Nuclear Information System (INIS)
Engl, Tobias; Makarević, Jasmina; Relja, Borna; Natsheh, Iyad; Müller, Iris; Beecken, Wolf-Dietrich; Jonas, Dietger; Blaheta, Roman A
2005-01-01
Tumor development remains one of the major obstacles following organ transplantation. Immunosuppressive drugs such as cyclosporine and tacrolimus directly contribute to enhanced malignancy, whereas the influence of the novel compound mycophenolate mofetil (MMF) on tumor cell dissemination has not been explored. We therefore investigated the adhesion capacity of colon, pancreas, prostate and kidney carcinoma cell lines to endothelium, as well as their beta1 integrin expression profile before and after MMF treatment. Tumor cell adhesion to endothelial cell monolayers was evaluated in the presence of 0.1 and 1 μM MMF and compared to unstimulated controls. beta1 integrin analysis included alpha1beta1 (CD49a), alpha2beta1 (CD49b), alpha3beta1 (CD49c), alpha4beta1 (CD49d), alpha5beta1 (CD49e), and alpha6beta1 (CD49f) receptors, and was carried out by reverse transcriptase-polymerase chain reaction, confocal microscopy and flow cytometry. Adhesion of the colon carcinoma cell line HT-29 was strongly reduced in the presence of 0.1 μM MMF. This effect was accompanied by down-regulation of alpha3beta1 and alpha6beta1 surface expression and of alpha3beta1 and alpha6beta1 coding mRNA. Adhesion of the prostate tumor cell line DU-145 was blocked dose-dependently by MMF. In contrast to MMF's effects on HT-29 cells, MMF dose-dependently up-regulated alpha1beta1, alpha2beta1, alpha3beta1, and alpha5beta1 on DU-145 tumor cell membranes. We conclude that MMF possesses distinct anti-tumoral properties, particularly in colon and prostate carcinoma cells. Adhesion blockage of HT-29 cells was due to the loss of alpha3beta1 and alpha6beta1 surface expression, which might contribute to a reduced invasive behaviour of this tumor entity. The enhancement of integrin beta1 subtypes observed in DU-145 cells possibly causes re-differentiation towards a low-invasive phenotype
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Lv, Jianwei [General Hospital of Tianjin Medical University, No. 154, Anshan Road, Heping District, Tianjin 300052 (China); Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin 300050 (China); Sun, Xiaolei; Ma, Jianxiong [Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin 300050 (China); Ma, Xinlong, E-mail: gengxiao502@163.com [General Hospital of Tianjin Medical University, No. 154, Anshan Road, Heping District, Tianjin 300052 (China); Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin 300050 (China); Zhang, Yang; Li, Fengbo; Li, Yanjun; Zhao, Zhihu [Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin 300050 (China)
2015-08-14
Schwann cells (SCs) play an essentially supportive role in the regeneration of injured peripheral nerve system (PNS). As Netrin-1 is crucial for the normal development of nervous system (NS) and can direct the process of damaged PNS regeneration, our study was designed to determine the role of Netrin-1 in RSC96 Schwann cells (an immortalized rat Schwann cell line) proliferation and migration. Our studies demonstrated that Netrin-1 had no effect on RSC96 cells proliferation, while significantly promoted RSC96 cells migration. The Netrin-1-induced RSC96 cells migration was significantly attenuated by inhibition of p38 and PI3K through pretreatment with SB203580 and LY294002 respectively, but not inhibition of MEK1/2 and JNK by U0126-EtOH and SP600125 individually. Treatment with Netrin-1 enhanced the phosphorylation of p38 and Akt. QRT-PCR indicated that Netrin-1 and only its receptors Unc5a, Unc5b and Neogenin were expressed in RSC96 cells, among which Unc5b expressed the most. And UNC5B protein was significantly increased after stimulated by Netrin-1. In conclusion, we show here that Netrin-1-enhanced SCs migration is mediated by activating p38 MAPK and PI3K-Akt signal cascades via receptor UNC5B, which suggests that Netrin-1 could serve as a new therapeutic strategy and has potential application value for PNS regeneration. - Highlights: • Netrin-1 attracts RSC96 Schwann cells migration in a dose dependent manner. • Netrin-1 induced Schwann cells migration is p38 and PI3K-Akt signaling dependent. • UNC5B may be dominant receptor mediating Netrin-1′ effect on RSC96 cells motility. • Netrin-1 may promote peripheral nerve repair by enhancing Schwann cells motility.
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Casimiro-Garcia, Agustin; Filzen, Gary F.; Flynn, Declan; Bigge, Christopher F.; Chen, Jing; Davis, Jo Ann; Dudley, Danette A.; Edmunds, Jeremy J.; Esmaeil, Nadia; Geyer, Andrew; Heemstra, Ronald J.; Jalaie, Mehran; Ohren, Jeffrey F.; Ostroski, Robert; Ellis, Teresa; Schaum, Robert P.; Stoner, Chad (Pfizer)
2013-03-07
Mining of an in-house collection of angiotensin II type 1 receptor antagonists to identify compounds with activity at the peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) revealed a new series of imidazo[4,5-b]pyridines 2 possessing activity at these two receptors. Early availability of the crystal structure of the lead compound 2a bound to the ligand binding domain of human PPAR{gamma} confirmed the mode of interaction of this scaffold to the nuclear receptor and assisted in the optimization of PPAR{gamma} activity. Among the new compounds, (S)-3-(5-(2-(1H-tetrazol-5-yl)phenyl)-2,3-dihydro-1H-inden-1-yl)-2-ethyl-5-isobutyl-7-methyl-3H-imidazo[4,5-b]pyridine (2l) was identified as a potent angiotensin II type I receptor blocker (IC{sub 50} = 1.6 nM) with partial PPAR{gamma} agonism (EC{sub 50} = 212 nM, 31% max) and oral bioavailability in rat. The dual pharmacology of 2l was demonstrated in animal models of hypertension (SHR) and insulin resistance (ZDF rat). In the SHR, 2l was highly efficacious in lowering blood pressure, while robust lowering of glucose and triglycerides was observed in the male ZDF rat.
Directory of Open Access Journals (Sweden)
Vilaiwan M. Fernandes
2014-12-01
Full Text Available During morphogenesis, extracellular signals trigger actomyosin contractility in subpopulations of cells to coordinate changes in cell shape. To illuminate the link between signaling-mediated tissue patterning and cytoskeletal remodeling, we study the progression of the morphogenetic furrow (MF, the wave of apical constriction that traverses the Drosophila eye imaginal disc preceding photoreceptor neurogenesis. Apical constriction depends on actomyosin contractility downstream of the Hedgehog (Hh and bone morphogenetic protein (BMP pathways. We identify a role for integrin adhesion receptors in MF progression. We show that Hh and BMP regulate integrin expression, the loss of which disrupts apical constriction and slows furrow progression; conversely, elevated integrins accelerate furrow progression. We present evidence that integrins regulate MF progression by promoting microtubule stabilization, since reducing microtubule stability rescues integrin-mediated furrow acceleration. Thus, integrins act as a genetic link between tissue-level signaling events and morphological change at the cellular level, leading to morphogenesis and neurogenesis in the eye.
Microglia P2Y13 Receptors Prevent Astrocyte Proliferation Mediated by P2Y1 Receptors
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Clara Quintas
2018-05-01
Full Text Available Cerebral inflammation is a common feature of several neurodegenerative diseases that requires a fine interplay between astrocytes and microglia to acquire appropriate phenotypes for an efficient response to neuronal damage. During brain inflammation, ATP is massively released into the extracellular medium and converted into ADP. Both nucleotides acting on P2 receptors, modulate astrogliosis through mechanisms involving microglia-astrocytes communication. In previous studies, primary cultures of astrocytes and co-cultures of astrocytes and microglia were used to investigate the influence of microglia on astroglial proliferation induced by ADPβS, a stable ADP analog. In astrocyte cultures, ADPβS increased cell proliferation through activation of P2Y1 and P2Y12 receptors, an effect abolished in co-cultures (of astrocytes with ∼12.5% microglia. The possibility that the loss of the ADPβS-mediated effect could have been caused by a microglia-induced degradation of ADPβS or by a preferential microglial localization of P2Y1 or P2Y12 receptors was excluded. Since ADPβS also activates P2Y13 receptors, the contribution of microglial P2Y13 receptors to prevent the proliferative effect of ADPβS in co-cultures was investigated. The results obtained indicate that P2Y13 receptors are low expressed in astrocytes and mainly expressed in microglia. Furthermore, in co-cultures, ADPβS induced astroglial proliferation in the presence of the selective P2Y13 antagonist MRS 2211 (3 μM and of the selective P2Y12 antagonist AR-C66096 (0.1 μM, suggesting that activation of microglial P2Y12 and P2Y13 receptors may induce the release of messengers that inhibit astroglial proliferation mediated by P2Y1,12 receptors. In this microglia-astrocyte paracrine communication, P2Y12 receptors exert opposite effects in astroglial proliferation as a result of its cellular localization: cooperating in astrocytes with P2Y1 receptors to directly stimulate proliferation and in
International Nuclear Information System (INIS)
Crowe, David L; Ohannessian, Arthur
2004-01-01
Integrin-extracellular matrix interactions activate signaling cascades such as mitogen activated protein kinases (MAPK). Integrin binding to extracellular matrix increases tyrosine phosphorylation of focal adhesion kinase (FAK). Inhibition of FAK activity by expression of its carboxyl terminus decreases cell motility, and cells from FAK deficient mice also show reduced migration. Paxillin is a focal adhesion protein which is also phosphorylated on tyrosine. FAK recruitment of paxillin to the cell membrane correlates with Shc phosphorylation and activation of MAPK. Decreased FAK expression inhibits papilloma formation in a mouse skin carcinogenesis model. We previously demonstrated that MAPK activation was required for growth factor induced in vitro migration and invasion by human squamous cell carcinoma (SCC) lines. Adapter protein recruitment to integrin subunits was examined by co-immunoprecipitation in SCC cells attached to type IV collagen or plastic. Stable clones overexpressing FAK or paxillin were created using the lipofection technique. Modified Boyden chambers were used for invasion assays. In the present study, we showed that FAK and paxillin but not Shc are recruited to the β1 integrin cytoplasmic domain following attachment of SCC cells to type IV collagen. Overexpression of either FAK or paxillin stimulated cancer cell migration on type IV collagen and invasion through reconstituted basement membrane which was dependent on MAPK activity. We concluded that recruitment of focal adhesion kinase and paxillin to β1 integrin promoted cancer cell migration via the mitogen activated protein kinase pathway
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Ohannessian Arthur
2004-05-01
Full Text Available Abstract Background Integrin-extracellular matrix interactions activate signaling cascades such as mitogen activated protein kinases (MAPK. Integrin binding to extracellular matrix increases tyrosine phosphorylation of focal adhesion kinase (FAK. Inhibition of FAK activity by expression of its carboxyl terminus decreases cell motility, and cells from FAK deficient mice also show reduced migration. Paxillin is a focal adhesion protein which is also phosphorylated on tyrosine. FAK recruitment of paxillin to the cell membrane correlates with Shc phosphorylation and activation of MAPK. Decreased FAK expression inhibits papilloma formation in a mouse skin carcinogenesis model. We previously demonstrated that MAPK activation was required for growth factor induced in vitro migration and invasion by human squamous cell carcinoma (SCC lines. Methods Adapter protein recruitment to integrin subunits was examined by co-immunoprecipitation in SCC cells attached to type IV collagen or plastic. Stable clones overexpressing FAK or paxillin were created using the lipofection technique. Modified Boyden chambers were used for invasion assays. Results In the present study, we showed that FAK and paxillin but not Shc are recruited to the β1 integrin cytoplasmic domain following attachment of SCC cells to type IV collagen. Overexpression of either FAK or paxillin stimulated cancer cell migration on type IV collagen and invasion through reconstituted basement membrane which was dependent on MAPK activity. Conclusions We concluded that recruitment of focal adhesion kinase and paxillin to β1 integrin promoted cancer cell migration via the mitogen activated protein kinase pathway.
Endothelial-Rac1 is not required for tumor angiogenesis unless alphavbeta3-integrin is absent.
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Gabriela D'Amico
2010-03-01
Full Text Available Endothelial cell migration is an essential aspect of tumor angiogenesis. Rac1 activity is needed for cell migration in vitro implying a requirement for this molecule in angiogenesis in vivo. However, a precise role for Rac1 in tumor angiogenesis has never been addressed. Here we show that depletion of endothelial Rac1 expression in adult mice, unexpectedly, has no effect on tumor growth or tumor angiogenesis. In addition, repression of Rac1 expression does not inhibit VEGF-mediated angiogenesis in vivo or ex vivo, nor does it affect chemotactic migratory responses to VEGF in 3-dimensions. In contrast, the requirement for Rac1 in tumor growth and angiogenesis becomes important when endothelial beta3-integrin levels are reduced or absent: the enhanced tumor growth, tumor angiogenesis and VEGF-mediated responses in beta3-null mice are all Rac1-dependent. These data indicate that in the presence of alphavbeta3-integrin Rac1 is not required for tumor angiogenesis.
Bellocchio, Luigi; Soria-Gómez, Edgar; Quarta, Carmelo; Metna-Laurent, Mathilde; Cardinal, Pierre; Binder, Elke; Cannich, Astrid; Delamarre, Anna; Häring, Martin; Martín-Fontecha, Mar; Vega, David; Leste-Lasserre, Thierry; Bartsch, Dusan; Monory, Krisztina; Lutz, Beat; Chaouloff, Francis; Pagotto, Uberto; Guzman, Manuel; Cota, Daniela; Marsicano, Giovanni
2013-01-01
Complex interactions between periphery and the brain regulate food intake in mammals. Cannabinoid type-1 (CB1) receptor antagonists are potent hypophagic agents, but the sites where this acute action is exerted and the underlying mechanisms are not fully elucidated. To dissect the mechanisms underlying the hypophagic effect of CB1 receptor blockade, we combined the acute injection of the CB1 receptor antagonist rimonabant with the use of conditional CB1-knockout mice, as well as with pharmacological modulation of different central and peripheral circuits. Fasting/refeeding experiments revealed that CB1 receptor signaling in many specific brain neurons is dispensable for the acute hypophagic effects of rimonabant. CB1 receptor antagonist-induced hypophagia was fully abolished by peripheral blockade of β-adrenergic transmission, suggesting that this effect is mediated by increased activity of the sympathetic nervous system. Consistently, we found that rimonabant increases gastrointestinal metabolism via increased peripheral β-adrenergic receptor signaling in peripheral organs, including the gastrointestinal tract. Blockade of both visceral afferents and glutamatergic transmission in the nucleus tractus solitarii abolished rimonabant-induced hypophagia. Importantly, these mechanisms were specifically triggered by lipid-deprivation, revealing a nutrient-specific component acutely regulated by CB1 receptor blockade. Finally, peripheral blockade of sympathetic neurotransmission also blunted central effects of CB1 receptor blockade, such as fear responses and anxiety-like behaviors. These data demonstrate that, independently of their site of origin, important effects of CB1 receptor blockade are expressed via activation of peripheral sympathetic activity. Thus, CB1 receptors modulate bidirectional circuits between the periphery and the brain to regulate feeding and other behaviors. PMID:23487769
p62 regulates CD40-mediated NFκB activation in macrophages through interaction with TRAF6
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Seibold, Kristina; Ehrenschwender, Martin, E-mail: martin.ehrenschwender@ukr.de
2015-08-14
CD40 is a member of the tumor necrosis factor (TNF) receptor family. Activation-induced recruitment of adapter proteins, so-called TNF-receptor-associated factors (TRAFs) to the cytoplasmic tail of CD40 triggers signaling cascades important in the immune system, but has also been associated with excessive inflammation in diseases such as atherosclerosis and rheumatoid arthritis. Especially, pro-inflammatory nuclear factor κB (NFκB) signaling emanating from CD40-associated TRAF6 appears to be a key pathogenic driving force. Consequently, targeting the CD40-TRAF6 interaction is emerging as a promising therapeutic strategy, but the underlying molecular machinery of this signaling axis is to date poorly understood. Here, we identified the multifunctional adaptor protein p62 as a critical regulator in CD40-mediated NFκB signaling via TRAF6. CD40 activation triggered formation of a TRAF6-p62 complex. Disturbing this interaction tremendously reduced CD40-mediated NFκB signaling in macrophages, while TRAF6-independent signaling pathways remained unaffected. This highlights p62 as a potential target in hyper-inflammatory, CD40-associated pathologies. - Highlights: • CD40 activation triggers interaction of the adapter protein TRAF6 with p62. • TRAF6-p62 interaction regulates CD40-mediated NFκB signaling in macrophages. • Defective TRAF6-p62 interaction reduces CD40-mediated NFκB activation in macrophages.
Moloney, G P; Martin, G R; Mathews, N; Milne, A; Hobbs, H; Dodsworth, S; Sang, P Y; Knight, C; Williams, M; Maxwell, M; Glen, R C
1999-07-15
The synthesis and vascular 5-HT(1B)-like receptor activity of a novel series of substituted 2, N-benzylcarboxamido-5-(2-ethyl-1-dioxoimidazolidinyl)-N, N-dimethyltryptamine derivatives are described. Modifications to the 5-ethylene-linked heterocycle and to substituents on the 2-benzylamide side chain have been explored. Several compounds were identified which exhibited affinity at the vascular 5-HT(1B)-like receptor of pK(B) > 7.0, up to 100-fold selectivity over alpha(1)-adrenoceptor affinity and 5-HT(2A) receptor affinity, and which exhibited a favorable pharmacokinetic profile. N-Benzyl-3-[2-(dimethylamino)ethyl]-5-[2-(4,4-dimethyl-2, 5-dioxo-1-imidazolidinyl)ethyl]-1H-indole-2-carboxamide (23) was identified as a highly potent, silent (as judged by the inability of angiotensin II to unmask 5-HT(1B)-like receptor-mediated agonist activity in the rabbit femoral artery), and competitive vascular 5-HT(1B)-like receptor antagonist with a plasma elimination half-life of approximately 4 h in dog plasma and with good oral bioavailability. The selectivity of compounds from this series for the vascular 5-HT(1B)-like receptors over other receptor subtypes is discussed as well as a proposed mode of binding to the receptor pharmacophore. It has been proposed that the aromatic ring of the 2, N-benzylcarboxamide group can occupy an aromatic binding site rather than the indole ring. The resulting conformation allows an amine-binding site to be occupied by the ethylamine nitrogen and a hydrogen-bonding site to be occupied by one of the hydantoin carbonyls. The electronic nature of the 2,N-benzylcarboxamide aromatic group as well as the size of substituents on this aromatic group is crucial for producing potent and selective antagonists. The structural requirement on the 3-ethylamine side chain incorporating the protonatable nitrogen is achieved by the bulky 2, N-benzylcarboxamide group and its close proximity to the 3-side chain.
Role of β1-Integrin in Colorectal Cancer: Case-Control Study
Oh, Bo-Young; Kim, Kwang Ho; Chung, Soon Sup; Hong, Kyoung Sook
2014-01-01
Purpose In the metastatic process, interactions between circulating tumor cells (CTCs) and the extracellular matrix or surrounding cells are required. β1-Integrin may mediate these interactions. The aim of this study was to investigate whether β1-integrin is associated with the detection of CTCs in colorectal cancer. Methods We enrolled 30 patients with colorectal cancer (experimental group) and 30 patients with benign diseases (control group). Blood samples were obtained from each group, carcinoembryonic antigen (CEA) mRNA for CTCs marker and β1-integrin mRNA levels were estimated by using reverse transcription-polymerase chain reaction, and the results were compared between the two groups. In the experimental group, preoperative results were compared with postoperative results for each marker. In addition, we analyzed the correlation between the expressions of β1-integrin and CEA. Results CEA mRNA was detected more frequently in colorectal cancer patients than in control patients (P = 0.008). CEA mRNA was significantly reduced after surgery in the colorectal cancer patients (P = 0.032). β1-Integrin mRNA was detected more in colorectal cancer patients than in the patients with benign diseases (P < 0.001). In colorectal cancer patients, expression of β1-integrin mRNA was detected more for advanced-stage cancer than for early-stage cancer (P = 0.033) and was significantly decreased after surgery (P < 0.001). In addition, expression of β1-integrin mRNA was significantly associated with that of CEA mRNA in colorectal cancer patients (P = 0.001). Conclusion In conclusion, β1-integrin is a potential factor for forming a prognosis following surgical resection in colorectal cancer patients. β1-Integrin may be a candidate for use as a marker for early detection of micrometastatic tumor cells and for monitoring the therapeutic response in colorectal cancer patients. PMID:24851215
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Stephan eMeinke
2013-01-01
Full Text Available 2B4 (CD244 and NK-T-B-antigen (NTB-A, CD352 are activating receptors on human NK cells and belong to the family of SLAM-related receptors. Engagement of these receptors leads to phosphorylation of their cytoplasmic tails and recruitment of the adapter proteins SAP and EAT-2. X-linked lymphoproliferative syndrome (XLP is a severe immunodeficiency that results from mutations in the SAP gene. 2B4 and NTB-A-mediated cytotoxicity are abrogated in XLP NK cells. To elucidate the molecular basis for this defect we analyzed early signaling events in SAP knockdown cells. Similar to XLP NK cells, knockdown of SAP in primary human NK cells leads to a reduction of 2B4 and NTB-A-mediated cytotoxicity. We found that early signaling events such as raft recruitment and receptor phosphorylation are not affected by the absence of SAP, indicating the defect in the absence of SAP is downstream of these events. In addition, knockdown of EAT-2 does not impair 2B4 or NTB-A-mediated cytotoxicity. Surprisingly, EAT-2 recruitment to both receptors is abrogated in the absence of SAP, revealing a novel cooperativity between these adapters.
Pernomian, Larissa; Pernomian, Laena; Gomes, Mayara S; da Silva, Carlos H T P
2015-12-15
The interplay between angiotensin AT1 receptors and MAS receptors relies on several inward regulatory mechanisms from renin-angiotensin system (RAS) including the functional crosstalk between angiotensin II and angiotensin-(1-7), the competitive AT1 antagonism exhibited by angiotensin-(1-7), the antagonist feature assigned to AT1/MAS heterodimerization on AT1 signaling and the AT1-mediated downregulation of angiotensin-converting enzyme 2 (ACE2). Recently, such interplay has acquired an important significance to RAS Pharmacology since a few studies have supporting strong evidences that MAS receptors mediate the effects elicited by AT1 antagonists. The present Perspective provides an overview of the regulatory mechanisms involving AT1 and MAS receptors, their significance to RAS Pharmacology and the future directions on the interplay between angiotensin receptors. Copyright © 2015 Elsevier B.V. All rights reserved.
Characterization of Laminin Binding Integrin Internalization in Prostate Cancer Cells†
Das, Lipsa; Anderson, Todd A.; Gard, Jaime M.C.; Sroka, Isis C.; Strautman, Stephanie R.; Nagle, Raymond B.; Morrissey, Colm; Knudsen, Beatrice S.; Cress, Anne E.
2017-01-01
Laminin binding integrins α6 (CD49f) and α3 (CD49c) are persistently but differentially expressed in prostate cancer (PCa). Integrin internalization is an important determinant of their cell surface expression and function. Using flow cytometry, and first order kinetic modelling, we quantitated the intrinsic internalization rates of integrin subunits in a single cycle of internalization. In PCa cell line DU145, α6 integrin internalized with a rate constant (kactual) of 3.25min−1, 3-fold faster than α3 integrin (1.0 min−1), 1.5-fold faster than the vitronectin binding αv integrin (CD51) (2.2 min−1), and significantly slower than the unrelated transferrin receptor (CD71) (15 min−1). Silencing of α3 integrin protein expression in DU145, PC3 and PC3B1 cells resulted in up to a 1.71-fold increase in kactual for α6 integrin. The internalized α6 integrin was targeted to early endosomes but not to lamp1 vesicles. Depletion of α3 integrin expression resulted in redistribution of α6β4 integrin to an observed cell-cell staining pattern that is consistent with a suprabasal distribution observed in epidermis and early PIN lesions in PCa. Depletion of α3 integrin increased cell migration by 1.8 fold, which was dependent on α6β1 integrin. Silencing of α6 integrin expression however, had no significant effect on the kactual of α3 integrin or its distribution in early endosomes. These results indicate that α3 and α6 integrins have significantly different internalization kinetics and that coordination exists between them for internalization. This article is protected by copyright. All rights reserved PMID:27509031
Characterization of Laminin Binding Integrin Internalization in Prostate Cancer Cells.
Das, Lipsa; Anderson, Todd A; Gard, Jaime M C; Sroka, Isis C; Strautman, Stephanie R; Nagle, Raymond B; Morrissey, Colm; Knudsen, Beatrice S; Cress, Anne E
2017-05-01
Laminin binding integrins α6 (CD49f) and α3 (CD49c) are persistently but differentially expressed in prostate cancer (PCa). Integrin internalization is an important determinant of their cell surface expression and function. Using flow cytometry, and first order kinetic modeling, we quantitated the intrinsic internalization rates of integrin subunits in a single cycle of internalization. In PCa cell line DU145, α6 integrin internalized with a rate constant (k actual ) of 3.25 min -1 , threefold faster than α3 integrin (1.0 min -1 ), 1.5-fold faster than the vitronectin binding αv integrin (CD51) (2.2 min -1 ), and significantly slower than the unrelated transferrin receptor (CD71) (15 min -1 ). Silencing of α3 integrin protein expression in DU145, PC3, and PC3B1 cells resulted in up to a 1.71-fold increase in k actual for α6 integrin. The internalized α6 integrin was targeted to early endosomes but not to lamp1 vesicles. Depletion of α3 integrin expression resulted in redistribution of α6β4 integrin to an observed cell-cell staining pattern that is consistent with a suprabasal distribution observed in epidermis and early PIN lesions in PCa. Depletion of α3 integrin increased cell migration by 1.8-fold, which was dependent on α6β1 integrin. Silencing of α6 integrin expression however, had no significant effect on the k actual of α3 integrin or its distribution in early endosomes. These results indicate that α3 and α6 integrins have significantly different internalization kinetics and that coordination exists between them for internalization. J. Cell. Biochem. 118: 1038-1049, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.
Mechanism of Integrim-Mediated Growth Control in Normal, Transformed, and Neoplastic Breast Cells
National Research Council Canada - National Science Library
Wayner, Elizabeth
1999-01-01
.... The primary cell adhesion receptors that mediate binding to extracellular matrix proteins are integrins Our data suggest that alpha 3 beta 1 and alpha 6 beta 4 are the primary integrins responsible...
Proteinase-activated receptors - mediators of early and delayed normal tissue radiation responses
International Nuclear Information System (INIS)
Hauer-Jensen, M.
2003-01-01
Proteinase-activated receptors (PARs) are G-protein coupled receptors that are activated by proteolytic exposure of a receptor-tethered ligand. The discovery of this receptor family represents one of the most intriguing recent developments in signal transduction. PARs are involved in the regulation of many normal and pathophysiological processes, notably inflammatory and fibroproliferative responses to injury. Preclinical studies performed in our laboratory suggest that proteinase-activated receptor-1 (PAR-1) plays a critical role in the mechanism of chronicity of radiation fibrosis, while proteinase-activated receptor-2 (PAR-2) may mediate important fibroproliferative responses in irradiated intestine. Specifically, activation of PAR-1 by thrombin, and PAR-2 by pancreatic trypsin and mast cell proteinases, appears to be involved in acute radiation-induced inflammation, as well as in subsequent extracellular matrix deposition, leading to the development of intestinal wall fibrosis and clinical complications. Pharmacological modulators of PAR-1 or PAR-2 expression or activation would be potentially useful as preventive or therapeutic agents in patients who receive radiation therapy, especially if blockade could be targeted to specific tissues or cellular compartments
Kim, Jung; Bilder, David; Neufeld, Thomas P
2018-01-15
Insulin resistance, the failure to activate insulin signaling in the presence of ligand, leads to metabolic diseases, including type 2 diabetes. Physical activity and mechanical stress have been shown to protect against insulin resistance, but the molecular mechanisms remain unclear. Here, we address this relationship in the Drosophila larval fat body, an insulin-sensitive organ analogous to vertebrate adipose tissue and livers. We found that insulin signaling in Drosophila fat body cells is abolished in the absence of physical activity and mechanical stress even when excess insulin is present. Physical movement is required for insulin sensitivity in both intact larvae and fat bodies cultured ex vivo. Interestingly, the insulin receptor and other downstream components are recruited to the plasma membrane in response to mechanical stress, and this membrane localization is rapidly lost upon disruption of larval or tissue movement. Sensing of mechanical stimuli is mediated in part by integrins, whose activation is necessary and sufficient for mechanical stress-dependent insulin signaling. Insulin resistance develops naturally during the transition from the active larval stage to the immotile pupal stage, suggesting that regulation of insulin sensitivity by mechanical stress may help coordinate developmental programming with metabolism. © 2018 Kim et al.; Published by Cold Spring Harbor Laboratory Press.
Directory of Open Access Journals (Sweden)
Mingsheng Cai
Full Text Available The innate immune response plays a critical role in the host defense against invading pathogens, and TLR2, a member of the Toll-like receptor (TLR family, has been implicated in the immune response and initiation of inflammatory cytokine secretion against several human viruses. Previous studies have demonstrated that infectious and ultraviolet-inactivated herpes simplex virus 1 (HSV-1 virions lead to the activation of nuclear factor kappa B (NF-κB and secretion of proinflammatory cytokines via TLR2. However, except for the envelope glycoprotein gH and gL, whether there are other determinants of HSV-1 responsible for TLR2 mediated biological effects is not known yet. Here, we demonstrated that the HSV-1-encoded envelope glycoprotein gB displays as molecular target recognized by TLR2. gB coimmunoprecipitated with TLR2, TLR1 and TLR6 in transfected and infected human embryonic kidney (HEK 293T cells. Treatment of TLR2-transfected HEK293T (HEK293T-TLR2 cells with purified gB results in the activation of NF-κB reporter, and this activation requires the recruitment of the adaptor molecules myeloid differentiation primary-response protein 88 (MyD88 and tumor necrosis factor receptor-associated factor 6 (TRAF6 but not CD14. Furthermore, activation of NF-κB was abrogated by anti-gB and anti-TLR2 blocking antibodies. In addition, the expression of interleukin-8 induced by gB was abrogated by the treatment of the human monocytic cell line THP-1 with anti-TLR2 blocking antibody or by the incubation of gB with anti-gB antibody. Taken together, these results indicate the importance and potency of HSV-1 gB as one of pathogen-associated molecular patterns (PAMPs molecule recognized by TLR2 with immediate kinetics.
Directory of Open Access Journals (Sweden)
G Anastasi
2009-06-01
Full Text Available Sarcoglycans are transmembrane proteins that seem to be functionally and pathologically as important as dystrophin. Sarcoglycans cluster together to form a complex, which is localized in the cell membrane of skeletal, cardiac, and smooth muscle. It has been proposed that the dystrophin-glycoprotein complex (DGC links the actin cytoskeleton with the extracellular matrix and the proper maintenance of this connection is thought to be crucial to the mechanical stability of the sarcolemma. The integrins are a family of heterodimeric cell surface receptors which play a crucial role in cell adhesion including cell-matrix and intracellular interactions and therefore are involved in various biological phenomena, including cell migration, and differentiation tissue repair. Sarcoglycans and integrins play a mechanical and signaling role stabilizing the systems during cycles of contraction and relaxation.Several studies suggested the possibility that integrins might play a role in muscle agrin signalling. On these basis, we performed an immunohistochemical analyzing sarcoglycans, integrins and agrin, on human skeletal muscle affected by sensitive-motor polyneuropathy, in order to better define the correlation between these proteins and neurogenic atrophy due to peripheral neuropathy. Our results showed the existence of a cascade mechanism which provoke a loss of regulatory effects of muscle activity on costameres, due to loss of muscle and neural agrin.This cascade mechanism could determine a quantitative modification of transmembrane receptors and loss of ?7B could be replaced and reinforced by enhanced expression of the ?7A integrin to restore muscle fiber viability. Second, it is possible that the reduced cycles of contraction and relaxation of muscle fibers, during muscular atrophy, provoke a loss of mechanical stresses transmitted over cell surface receptors that physically couple the cytoskeleton to extracellular matrix. Consequently, these mechanical
Microvesicle transfer of kinin B1-receptors is a novel inflammatory mechanism in vasculitis.
Kahn, Robin; Mossberg, Maria; Ståhl, Anne-Lie; Johansson, Karl; Lopatko Lindman, Ingrid; Heijl, Caroline; Segelmark, Mårten; Mörgelin, Matthias; Leeb-Lundberg, L M Fredrik; Karpman, Diana
2017-01-01
During vasculitis, activation of the kinin system induces inflammation, whereby the kinin B1-receptor is expressed and activated after ligand binding. Additionally, activated blood cells release microvesicles into the circulation. Here we determined whether leukocyte-derived microvesicles bear B1-kinin receptors during vasculitis, and if microvesicles transfer functional B1-receptors to recipient cells, thus promoting inflammation. By flow cytometry, plasma from patients with vasculitis were found to contain high levels of leukocyte-derived microvesicles bearing B1-receptors. Importantly, renal biopsies from two patients with vasculitis showed leukocyte-derived microvesicles bearing B1-receptors docking on glomerular endothelial cells providing in vivo relevance. Microvesicles derived from B1-receptor-transfected human embryonic kidney cells transferred B1-receptors to wild-type human embryonic kidney cells, lacking the receptor, and to glomerular endothelial cells. The transferred B1-receptors induced calcium influx after B1-receptor agonist stimulation: a response abrogated by a specific B1-receptor antagonist. Microvesicles derived from neutrophils also transferred B1-receptors to wild-type human embryonic kidney cells and induced calcium influx after stimulation. Thus, we found a novel mechanism by which microvesicles transfer functional receptors and promote kinin-associated inflammation. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.
Yang, Dayun; Lü, Xiaoying; Hong, Ying; Xi, Tingfei; Zhang, Deyuan
2013-07-01
To explore molecular mechanism of mediation of adsorbed proteins to cell adhesion and growth on biomaterials, this study examined endothelial cell adhesion, morphology and viability on bare and titanium nitride (TiN) coated nickel titanium (NiTi) alloys and chitosan film firstly, and then identified the type and amount of serum proteins adsorbed on the three surfaces by proteomic technology. Subsequently, the mediation role of the identified proteins to cell adhesion and growth was investigated with bioinformatics analyses, and further confirmed by a series of cellular and molecular biological experiments. Results showed that the type and amount of adsorbed serum proteins associated with cell adhesion and growth was obviously higher on the alloys than on the chitosan film, and these proteins mediated endothelial cell adhesion and growth on the alloys via four ways. First, proteins such as adiponectin in the adsorbed protein layer bound with cell surface receptors to generate signal transduction, which activated cell surface integrins through increasing intracellular calcium level. Another way, thrombospondin 1 in the adsorbed protein layer promoted TGF-β signaling pathway activation and enhanced integrins expression. The third, RGD sequence containing proteins such as fibronectin 1, vitronectin and thrombospondin 1 in the adsorbed protein layer bound with activated integrins to activate focal adhesion pathway, increased focal adhesion formation and actin cytoskeleton organization and mediated cell adhesion and spreading. In addition, the activated focal adhesion pathway promoted the expression of cell growth related genes and resulted in cell proliferation. The fourth route, coagulation factor II (F2) and fibronectin 1 in the adsorbed protein layer bound with cell surface F2 receptor and integrin, activated regulation of actin cytoskeleton pathway and regulated actin cytoskeleton organization. Copyright © 2013 Elsevier Ltd. All rights reserved.
Yung, Bryan S; Brand, Cameron S; Xiang, Sunny Y; Gray, Charles B B; Means, Christopher K; Rosen, Hugh; Chun, Jerold; Purcell, Nicole H; Brown, Joan Heller; Miyamoto, Shigeki
2017-02-01
Sphingosine-1-phosphate (S1P), a bioactive lysophospholipid, is generated and released at sites of tissue injury in the heart and can act on S1P 1 , S1P 2 , and S1P 3 receptor subtypes to affect cardiovascular responses. We established that S1P causes little phosphoinositide hydrolysis and does not induce hypertrophy indicating that it does not cause receptor coupling to G q . We previously demonstrated that S1P confers cardioprotection against ischemia/reperfusion by activating RhoA and its downstream effector PKD. The S1P receptor subtypes and G proteins that regulate RhoA activation and downstream responses in the heart have not been determined. Using siRNA or pertussis toxin to inhibit different G proteins in NRVMs we established that S1P regulates RhoA activation through Gα 13 but not Gα 12 , Gα q , or Gα i . Knockdown of the three major S1P receptors using siRNA demonstrated a requirement for S1P 3 in RhoA activation and subsequent phosphorylation of PKD, and this was confirmed in studies using isolated hearts from S1P 3 knockout (KO) mice. S1P treatment reduced infarct size induced by ischemia/reperfusion in Langendorff perfused wild-type (WT) hearts and this protection was abolished in the S1P 3 KO mouse heart. CYM-51736, an S1P 3 -specific agonist, also decreased infarct size after ischemia/reperfusion to a degree similar to that achieved by S1P. The finding that S1P 3 receptor- and Gα 13 -mediated RhoA activation is responsible for protection against ischemia/reperfusion suggests that selective targeting of S1P 3 receptors could provide therapeutic benefits in ischemic heart disease. Copyright © 2016 Elsevier Ltd. All rights reserved.
Torilin Inhibits Inflammation by Limiting TAK1-Mediated MAP Kinase and NF-κB Activation
Directory of Open Access Journals (Sweden)
Mehari Endale
2017-01-01
Full Text Available Torilin, a sesquiterpene isolated from the fruits of Torilis japonica, has shown antimicrobial, anticancer, and anti-inflammatory properties. However, data on the mechanism of torilin action against inflammation is limited. This study aimed at determining the anti-inflammatory property of torilin in LPS-induced inflammation using in vitro model of inflammation. We examined torilin’s effect on expression levels of inflammatory mediators and cytokines in LPS-stimulated RAW 264.7 macrophages. The involvement of NF-kB and AP-1, MAP kinases, and adaptor proteins were assessed. Torilin strongly inhibited LPS-induced NO release, iNOS, PGE2, COX-2, NF-α, IL-1β, IL-6, and GM-CSF gene and protein expressions. In addition, MAPKs were also suppressed by torilin pretreatment. Involvement of ERK1/2, P38MAPK, and JNK1/2 was further confirmed by PD98059, SB203580, and SP600125 mediated suppression of iNOS and COX-2 proteins. Furthermore, torilin attenuated NF-kB and AP-1 translocation, DNA binding, and reporter gene transcription. Interestingly, torilin inhibited TAK1 kinase activation with the subsequent suppression of MAPK-mediated JNK, p38, ERK1/2, and AP-1 (ATF-2 and c-jun activation and IKK-mediated I-κBα degradation, p65/p50 activation, and translocation. Together, the results revealed the suppression of NF-κB and AP-1 regulated inflammatory mediator and cytokine expressions, suggesting the test compound’s potential as a candidate anti-inflammatory agent.
International Nuclear Information System (INIS)
Boettiger, D; Wehrle-Haller, B
2010-01-01
The measurement of cell adhesion using single cell force spectroscopy methods was compared with earlier methods for measuring cell adhesion. This comparison provided a means and rationale for separating components of the measurement retract curve that were due to interactions between the substrate and the glycocalyx, and interactions that were due to cell surface integrins binding to a substrate-bound ligand. The glycocalyx adhesion was characterized by multiple jumps with dispersed jump sizes that extended from 5 to 30 μm from the origin. The integrin mediated adhesion was represented by the F max (maximum detachment force), was generally within the first 5 μm and commonly detached with a single rupture cascade. The integrin peak (F max ) increases with time and the rate of increase shows large cell to cell variability with a peak ∼ 50 nN s -1 and an average rate of increase of 75 pN s -1 . This is a measure of the rate of increase in the number of adhesive integrin-ligand bonds/cell as a function of contact time.
Mechanical control of cyclic AMP signalling and gene transcription through integrins
Meyer, C. J.; Alenghat, F. J.; Rim, P.; Fong, J. H.; Fabry, B.; Ingber, D. E.
2000-01-01
This study was carried out to discriminate between two alternative hypotheses as to how cells sense mechanical forces and transduce them into changes in gene transcription. Do cells sense mechanical signals through generalized membrane distortion or through specific transmembrane receptors, such as integrins? Here we show that mechanical stresses applied to the cell surface alter the cyclic AMP signalling cascade and downstream gene transcription by modulating local release of signals generated by activated integrin receptors in a G-protein-dependent manner, whereas distortion of integrins in the absence of receptor occupancy has no effect.
Protein tyrosine phosphatase 1B (PTP1B) is dispensable for IgE-mediated cutaneous reaction in vivo.
Yang, Ting; Xie, Zhongping; Li, Hua; Yue, Lei; Pang, Zheng; MacNeil, Adam J; Tremblay, Michel L; Tang, Jin-Tian; Lin, Tong-Jun
2016-01-01
Mast cells play a critical role in allergic reactions. The cross-linking of FcεRI-bound IgE with multivalent antigen initiates a cascade of signaling events leading to mast cell activation. It has been well-recognized that cross linking of FcεRI mediates tyrosine phosphorylation. However, the mechanism involved in tyrosine dephosphorylation in mast cells is less clear. Here we demonstrated that protein tyrosine phosphatase 1B (PTP1B)-deficient mast cells showed increased IgE-mediated phosphorylation of the signal transducer and activator of transcription 5 (STAT5) and enhanced production of CCL9 (MIP-1γ) and IL-6 in IgE-mediated mast cells activation in vitro. However, IgE-mediated calcium mobilization, β-hexaosaminidase release (degranulation), and phosphorylation of IκB and MAP kinases were not affected by PTP1B deficiency. Furthermore, PTP1B deficient mice showed normal IgE-dependent passive cutaneous anaphylaxis and late phase cutaneous reactions in vivo. Thus, PTP1B specifically regulates IgE-mediated STAT5 pathway, but is redundant in influencing mast cell function in vivo. Copyright © 2016 Elsevier Inc. All rights reserved.
Directory of Open Access Journals (Sweden)
Lisa Salazar
Full Text Available Cancer is a major public health problem worldwide. In the United States alone, 1 in 4 deaths is due to cancer and for 2013 a total of 1,660,290 new cancer cases and 580,350 cancer-related deaths are projected. Comprehensive profiling of multiple cancer genomes has revealed a highly complex genetic landscape in which a large number of altered genes, varying from tumor to tumor, impact core biological pathways and processes. This has implications for therapeutic targeting of signaling networks in the development of treatments for specific cancers. The NFκB transcription factor is constitutively active in a number of hematologic and solid tumors, and many signaling pathways implicated in cancer are likely connected to NFκB activation. A critical mediator of NFκB activity is TGFβ-activated kinase 1 (TAK1. Here, we identify TAK1 as a novel interacting protein and target of fibroblast growth factor receptor 3 (FGFR3 tyrosine kinase activity. We further demonstrate that activating mutations in FGFR3 associated with both multiple myeloma and bladder cancer can modulate expression of genes that regulate NFκB signaling, and promote both NFκB transcriptional activity and cell adhesion in a manner dependent on TAK1 expression in both cancer cell types. Our findings suggest TAK1 as a potential therapeutic target for FGFR3-associated cancers, and other malignancies in which TAK1 contributes to constitutive NFκB activation.
Essential role of integrin-linked kinase in regulation of phagocytosis in keratinocytes.
Sayedyahossein, Samar; Nini, Lylia; Irvine, Timothy S; Dagnino, Lina
2012-10-01
Phagocytic melanosome uptake by epidermal keratinocytes is a central protective mechanism against damage induced by ultraviolet radiation. Phagocytosis requires formation of pseudopodia via actin cytoskeleton rearrangements. Integrin-linked kinase (ILK) is an important modulator of actin cytoskeletal dynamics. We have examined the role of ILK in regulation of phagocytosis, using epidermal keratinocytes isolated from mice with epidermis-restricted Ilk gene inactivation. ILK-deficient cells exhibited severely impaired capacity to engulf fluorescent microspheres in response to stimulation of the keratinocyte growth factor (KGF) receptor or the protease-activated receptor-2. KGF induced ERK phosphorylation in ILK-expressing and ILK-deficient cells, suggesting that ILK is not essential for KGF receptor signaling. In contrast, KGF promoted activation of Rac1 and formation of pseudopodia in ILK-expressing, but not in ILK-deficient cells. Rac1-deficient keratinocytes also showed substantially impaired phagocytic ability, underlining the importance of ILK-dependent Rac1 function for particle engulfment. Finally, cross-modulation of KGF receptors by integrins may be another important element, as integrin β1-deficient keratinocytes also fail to show significant phagocytosis in response to KGF. Thus, we have identified a novel signaling pathway essential for phagocytosis in keratinocytes, which involves ILK-dependent activation of Rac1 in response to KGF, resulting in the formation of pseudopodia and particle uptake.
The CD157-integrin partnership controls transendothelial migration and adhesion of human monocytes.
Lo Buono, Nicola; Parrotta, Rossella; Morone, Simona; Bovino, Paola; Nacci, Giulia; Ortolan, Erika; Horenstein, Alberto L; Inzhutova, Alona; Ferrero, Enza; Funaro, Ada
2011-05-27
CD157, a member of the CD38 gene family, is an NAD-metabolizing ectoenzyme and a signaling molecule whose role in polarization, migration, and diapedesis of human granulocytes has been documented; however, the molecular events underpinning this role remain to be elucidated. This study focused on the role exerted by CD157 in monocyte migration across the endothelial lining and adhesion to extracellular matrix proteins. The results demonstrated that anti-CD157 antibodies block monocyte transmigration and adhesion to fibronectin and fibrinogen but that CD157 cross-linking is sufficient to overcome the block, suggesting an active signaling role for the molecule. Consistent with this is the observation that CD157 is prevalently located within the detergent-resistant membrane microdomains to which, upon clustering, it promotes the recruitment of β(1) and β(2) integrin, which, in turn, leads to the formation of a multimolecular complex favoring signal transduction. This functional cross-talk with integrins allows CD157 to act as a receptor despite its intrinsic structural inability to do so on its own. Intracellular signals mediated by CD157 rely on the integrin/Src/FAK (focal adhesion kinase) pathway, resulting in increased activity of the MAPK/ERK1/2 and the PI3K/Akt downstream signaling pathways, which are crucial in the control of monocyte transendothelial migration. Collectively, these findings indicate that CD157 acts as a molecular organizer of signaling-competent membrane microdomains and that it forms part of a larger molecular machine ruled by integrins. The CD157-integrin partnership provides optimal adhesion and transmigration of human monocytes.
Dias, Jenny Pena; Gariépy, Helaine De Brito; Ongali, Brice; Couture, Réjean
2015-07-01
Kinin B1 receptor (B1R) is virtually absent under physiological condition, yet it is highly expressed in models of diabetes mellitus. This study aims at determining: (1) whether B1R is induced in the brain of insulin-resistant rat through the oxidative stress; (2) the consequence of B1R activation on stereotypic nocifensive behavior; (3) the role of downstream putative mediators in B1R-induced behavioral activity. Sprague-Dawley rats were fed with 10% D-glucose in their drinking water or tap water (controls) for 4 or 12 weeks, combined either with a standard chow diet or a diet enriched with α-lipoic acid (1 g/kg feed) for 4 weeks. The distribution and density of brain B1R binding sites were assessed by autoradiography. Behavioral activity evoked by i.c.v. injection of the B1R agonist Sar-[D-Phe(8)]-des-Arg(9)-BK (10 μg) was measured before and after i.c.v. treatments with selective antagonists (10 μg) for kinin B1 (R-715, SSR240612), tachykinin NK1 (RP-67580) and glutamate NMDA (DL-AP5) receptors or with the inhibitor of NOS (L-NNA). Results showed significant increases of B1R binding sites in various brain areas of glucose-fed rats that could be prevented by the diet containing α-lipoic acid. The B1R agonist elicited head scratching, grooming, sniffing, rearing, digging, licking, face washing, wet dog shake, teeth chattering and biting in glucose-fed rats, which were absent after treatment with α-lipoic acid or antagonists/inhibitors. Data suggest that kinin B1R is upregulated by the oxidative stress in the brain of insulin-resistant rats and its activation causes stereotypic nocifensive behavior through the release of substance P, glutamate and NO. Copyright © 2015 Elsevier Inc. All rights reserved.
Effect of integrin α5β1 inhibition on SDF-l/CXCR4- mediated choroidal neovascularization
Directory of Open Access Journals (Sweden)
Yang Lyu
2018-05-01
Full Text Available AIM: To investigate the roles of integrins in choroidal neovascularization (CNV and their associations with the stromal cell-derived factor-1 (SDF-1/CXCR4 axis. METHODS: CNV lesions were induced in mice using laser photocoagulation. After CNV induction, all animals were randomly assigned to: control, SDF-1, SDF-1+age-related macular degeneration (AMD 3100 (CXCR4 inhibitor, and SDF-1+ATN161 (integrin α5β1 inhibitor groups; their effects on CNV progression were observed using hematoxylin eosin (HE staining, fundus fluorescein angiography (FFA grading and optical coherence tomography (OCT, and their effects on CXCR4/integrin α5 expression were evaluated using Western blot and double immunofluorescence staining. Hypoxia-exposed endothelial cells (ECs were used to simulate CNV in vitro, they were treated with SDF-1, combined with CXCR4 siRNA/AMD3100 or ATN161, and expression of integrin α5, cell migration and tube formation were analyzed. RESULTS: Integrin subunit α5 increased at 3rd and 7th day and decreased at 14th day in CNV mice, with no significant change of β1-integrin. CXCR4 expression in CNV mice had persistent increase within 14d after induction. SDF-1 treatment significantly promoted the CNV progression during 3-14d. The mean CNV length in AMD3100 and ATN161 group at day 7 was 270.13 and 264.23 μm in HE images, significantly lower than the mean length in SDF-1 (345.70 μm group. AMD3100 and ATN161 also significantly reduced thickness and leakage of CNV induced by SDF-1. Mean integrin α5 positive area in SDF-1 group reached 2.31×104 μm2, significantly higher than control (1.25×104 μm2, which decreased to 1.78×104 μm2 after AMD3100 treatment. About 61.36% of ECs in CNV lesions expressed α5 in SDF-1 group, which significantly decreased to 43.12% after AMD3100 treatment. In vitro, integrin α5 peaked by 6 folds after 6h of hypoxia exposure and CXCR4 gradually increased by up to 2.3 folds after 24h of hypoxia. Approximately 25
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Moitza Principe
2017-01-01
Full Text Available Abstract Background We have previously shown that in pancreatic ductal adenocarcinoma (PDA cells, the glycolytic enzyme alpha-enolase (ENO1 also acts as a plasminogen receptor and promotes invasion and metastasis formation. Moreover, ENO1 silencing in PDA cells induces oxidative stress, senescence and profoundly modifies PDA cell metabolism. Although anti-ENO1 antibody inhibits PDA cell migration and invasion, little is known about the role of ENO1 in regulating cell-cell and cell-matrix contacts. We therefore investigated the effect of ENO1 silencing on the modulation of cell morphology, adhesion to matrix substrates, cell invasiveness, and metastatic ability. Methods The membrane and cytoskeleton modifications that occurred in ENO1-silenced (shENO1 PDA cells were investigated by a combination of confocal microscopy and atomic force microscopy (AFM. The effect of ENO1 silencing was then evaluated by phenotypic and functional experiments to identify the role of ENO1 in adhesion, migration, and invasion, as well as in senescence and apoptosis. The experimental results were then validated in a mouse model. Results We observed a significant increase in the roughness of the cell membrane due to ENO1 silencing, a feature associated with an impaired ability to migrate and invade, along with a significant downregulation of proteins involved in cell-cell and cell-matrix adhesion, including alpha v/beta 3 integrin in shENO1 PDA cells. These changes impaired the ability of shENO1 cells to adhere to Collagen I and IV and Fibronectin and caused an increase in RGD-independent adhesion to vitronectin (VN via urokinase plasminogen activator receptor (uPAR. Binding of uPAR to VN triggers integrin-mediated signals, which result in ERK1-2 and RAC activation, accumulation of ROS, and senescence. In shENO1 cancer cells, the use of an anti-uPAR antibody caused significant reduction of ROS production and senescence. Overall, a decrease of in vitro and in vivo cell
DEFF Research Database (Denmark)
Campos, Lia S; Leone, Dino P; Relvas, Joao B
2004-01-01
, signalling is required for neural stem cell maintenance, as assessed by neurosphere formation, and inhibition or genetic ablation of beta1 integrin using cre/lox technology reduces the level of MAPK activity. We conclude that integrins are therefore an important part of the signalling mechanisms that control......The emerging evidence that stem cells develop in specialised niches highlights the potential role of environmental factors in their regulation. Here we examine the role of beta1 integrin/extracellular matrix interactions in neural stem cells. We find high levels of beta1 integrin expression...... in the stem-cell containing regions of the embryonic CNS, with associated expression of the laminin alpha2 chain. Expression levels of laminin alpha2 are reduced in the postnatal CNS, but a population of cells expressing high levels of beta1 remains. Using neurospheres - aggregate cultures, derived from...
Inhibition of PTP1B Restores IRS1-Mediated Hepatic Insulin Signaling in IRS2-Deficient Mice
González-Rodríguez, Águeda; Gutierrez, Jose A. Mas; Sanz-González, Silvia; Ros, Manuel; Burks, Deborah J.; Valverde, Ángela M.
2010-01-01
OBJECTIVE Mice with complete deletion of insulin receptor substrate 2 (IRS2) develop hyperglycemia, impaired hepatic insulin signaling, and elevated gluconeogenesis, whereas mice deficient for protein tyrosine phosphatase (PTP)1B display an opposing hepatic phenotype characterized by increased sensitivity to insulin. To define the relationship between these two signaling pathways in the regulation of liver metabolism, we used genetic and pharmacological approaches to study the effects of inhibiting PTP1B on hepatic insulin signaling and expression of gluconeogenic enzymes in IRS2−/− mice. RESEARCH DESIGN AND METHODS We analyzed glucose homeostasis and insulin signaling in liver and isolated hepatocytes from IRS2−/− and IRS2−/−/PTP1B−/− mice. Additionally, hepatic insulin signaling was assessed in control and IRS2−/− mice treated with resveratrol, an antioxidant present in red wine. RESULTS In livers of hyperglycemic IRS2−/− mice, the expression levels of PTP1B and its association with the insulin receptor (IR) were increased. The absence of PTP1B in the double-mutant mice restored hepatic IRS1-mediated phosphatidylinositol (PI) 3-kinase/Akt/Foxo1 signaling. Moreover, resveratrol treatment of hyperglycemic IRS2−/− mice decreased hepatic PTP1B mRNA and inhibited PTP1B activity, thereby restoring IRS1-mediated PI 3-kinase/Akt/Foxo1 signaling and peripheral insulin sensitivity. CONCLUSIONS By regulating the phosphorylation state of IR, PTB1B determines sensitivity to insulin in liver and exerts a unique role in the interplay between IRS1 and IRS2 in the modulation of hepatic insulin action. PMID:20028942
Directory of Open Access Journals (Sweden)
Maria B. R. Piva
2017-09-01
Full Text Available Background: integrins have been associated with the development of chemotherapy resistant tumour cells, mostly those of hematopoietic origin, by mediating the binding to the extracellular matrix. The relevance for solid tumour cells and the underlying mechanisms remain elusive. Methods: using MTT assays, we detected the loss in cisplatin sensitivity of human MV3 melanoma cells upon integrin activation. Underlying cellular pathways were evaluated by flow cytometry. A crosstalk between integrin activation and the canonical wnt signalling pathway was tested by measuring β-catenin activity. Results: MV3 cells display a higher resistance against cisplatin cytotoxicity when cellular integrins were activated by manganese or collagen. Proteome profiler array showed a deregulation of the integrin expression pattern by cisplatin. Integrin activation by manganese induces the phosphorylation of PI3K/AKT. The inhibition of PI3K using BEZ235 strongly increases cell sensitivity to cisplatin, blocking manganese and collagen effects. PI3K/AKT activates wnt signalling by blocking Gsk3-β, which was confirmed by β-catenin up-regulation and nuclear localization. Integrins did not affect E-cadherin expression levels, thus endothelial to mesenchymal transition (EMT can be excluded. Conclusion: This is the first report on an integrin/wnt signalling activation axis addressing the consequences for chemotherapy sensitiveness of melanoma cells, which thus offers novel therapeutic targets for approaches to interfere with chemoresistance.
Bjerregaard, Nils; Bøtkjær, Kenneth A; Helsen, Nicky; Andreasen, Peter A; Dupont, Daniel M
2015-07-01
Recombinant tissue-type plasminogen activator (tPA, trade name Alteplase), currently the only drug approved by the US Food and Drug Administration and the European Medicines Agency for the treatment of cerebral ischaemic stroke, has been implicated in a number of adverse effects reportedly mediated by interactions with the low-density lipoprotein (LDL) family receptors, including neuronal cell death and an increased risk of cerebral haemorrhage. The tissue-type plasminogen activator is the principal initiator of thrombolysis in human physiology, an effect that is mediated directly via localised activation of the plasmin zymogen plasminogen at the surface of fibrin clots in the vascular lumen. Here, we sought to identify a ligand to tPA capable of inhibiting the relevant LDL family receptors without interfering with the fibrinolytic activity of tPA. Systematic evolution of ligands by exponential enrichment (SELEX) was employed to isolate tPA-binding RNA aptamers, which were characterised in biochemical assays of tPA association to low density lipoprotein receptor-related protein-1 (LRP-1, an LDL receptor family member); tPA-mediated in vitro and ex vivo clot lysis; and tPA-mediated plasminogen activation in the absence and presence of a stimulating soluble fibrin fragment. Two aptamers, K18 and K32, had minimal effects on clot lysis, but were able to efficiently inhibit tPA-LRP-1 association and LDL receptor family-mediated endocytosis in human vascular endothelial cells and astrocytes. These observations suggest that coadministration alongside tPA may be a viable strategy to improve the safety of thrombolytic treatment of cerebral ischaemic stroke by restricting tPA activity to the vascular lumen.
Absence of integrin alpha 7 causes a novel form of muscular dystrophy.
Mayer, U; Saher, G; Fässler, R; Bornemann, A; Echtermeyer, F; von der Mark, H; Miosge, N; Pöschl, E; von der Mark, K
1997-11-01
Integrin alpha 7 beta 1 is a specific cellular receptor for the basement membrane protein laminin-1 (refs 1,2), as well as for the laminin isoforms -2 and -4 (ref. 3). The alpha 7 subunit is expressed mainly in skeletal and cardiac muscle and has been suggested to be involved in differentiation and migration processes during myogenesis. Three cytoplasmic and two extracellular splice variants that have been described are developmentally regulated and expressed in different sites in the muscle. In adult muscle, the alpha 7A and alpha 7B subunits are concentrated in myotendinous junctions but can also be detected in neuromuscular junctions and along the sarcolemmal membrane. To study the potential involvement of alpha 7 integrin, during myogenesis and its role in muscle integrity and function, we generated a null allele of the alpha 7 gene (Itga7) in the germline of mice by homologous recombination in embryonic stem (ES) cells. Surprisingly, mice homozygous for the mutation are viable and fertile, indicating that the alpha 7 beta 1 integrin is not essential for myogenesis. However, histological analysis of skeletal muscle revealed typical symptoms of a progressive muscular dystrophy starting soon after birth, but with a distinct variability in different muscle types. The observed histopathological changes strongly indicate an impairment of function of the myotendinous junctions. These findings demonstrate that alpha 7 beta 1 integrin represents an indispensable linkage between the muscle fibre and the extracellular matrix that is independent of the dystrophin-dystroglycan complex-mediated interaction of the cytoskeleton with the muscle basement membrane.
Yoshiga, Daigo; Sato, Naoichi; Torisu, Takehiro; Mori, Hiroyuki; Yoshida, Ryoko; Nakamura, Seiji; Takaesu, Giichi; Kobayashi, Takashi; Yoshimura, Akihiko
2007-05-01
Adipocyte differentiation is regulated by insulin and IGF-I, which transmit signals by activating their receptor tyrosine kinase. SH2-B is an adaptor protein containing pleckstrin homology and Src homology 2 (SH2) domains that have been implicated in insulin and IGF-I receptor signaling. In this study, we found a strong link between SH2-B levels and adipogenesis. The fat mass and expression of adipogenic genes including peroxisome proliferator-activated receptor gamma (PPARgamma) were reduced in white adipose tissue of SH2-B-/- mice. Reduced adipocyte differentiation of SH2-B-deficient mouse embryonic fibroblasts (MEFs) was observed in response to insulin and dexamethasone, whereas retroviral SH2-B overexpression enhanced differentiation of 3T3-L1 preadipocytes to adipocytes. SH2-B overexpression enhanced mRNA level of PPARgamma in 3T3-L1 cells, whereas PPARgamma levels were reduced in SH2-B-deficient MEFs in response to insulin. SH2-B-mediated up-regulation of PPARgamma mRNA was blocked by a phosphatidylinositol 3-kinase inhibitor, but not by a MAPK kinase inhibitor. Insulin-induced Akt activation and the phosphorylation of forkhead transcription factor (FKHR/Foxo1), a negative regulator of PPARgamma transcription, were up-regulated by SH2-B overexpression, but reduced in SH2-B-deficient MEFs. These data indicate that SH2-B is a key regulator of adipogenesis both in vivo and in vitro by regulating the insulin/IGF-I receptor-Akt-Foxo1-PPARgamma pathway.
Bolduc, Gilles R; Madoff, Lawrence C
2007-12-01
Group B Streptococcus (GBS) is the leading cause of bacterial pneumonia, sepsis and meningitis among neonates and a cause of morbidity among pregnant women and immunocompromised adults. GBS epithelial cell invasion is associated with expression of alpha C protein (ACP). Loss of ACP expression results in a decrease in GBS internalization and translocation across human cervical epithelial cells (ME180). Soluble ACP and its 170 amino acid N-terminal region (NtACP), but not the repeat protein RR', bind to ME180 cells and reduce internalization of wild-type GBS to levels obtained with an ACP-deficient isogenic mutant. In the current study, ACP colocalized with alpha(1)beta(1)-integrin, resulting in integrin clustering as determined by laser scanning confocal microscopy. NtACP contains two structural domains, D1 and D2. D1 is structurally similar to fibronectin's integrin-binding region (FnIII10). D1's (KT)D146 motif is structurally similar to the FnIII10 (RG)D1495 integrin-binding motif, suggesting that ACP binds alpha(1)beta(1)-integrin via the D1 domain. The (KT)D146A mutation within soluble NtACP reduced its ability to bind alpha(1)beta(1)-integrin and inhibit GBS internalization within ME180 cells. Thus ACP binding to human epithelial cell integrins appears to contribute to GBS internalization within epithelial cells.
DEFF Research Database (Denmark)
Iba, K; Albrechtsen, R; Gilpin, B
2000-01-01
The ADAMs (a disintegrin and metalloprotease) family of proteins is involved in a variety of cellular interactions, including cell adhesion and ecto- domain shedding. Here we show that ADAM 12 binds to cell surface syndecans. Three forms of recombinant ADAM 12 were used in these experiments......-dependent manner attach to ADAM 12 via members of the syndecan family. After binding to syndecans, mesenchymal cells spread and form focal adhesions and actin stress fibers. Integrin beta1 was responsible for cell spreading because function-blocking monoclonal antibodies completely inhibited cell spreading......, and chondroblasts lacking beta1 integrin attached but did not spread. These data suggest that mesenchymal cells use syndecans as the initial receptor for the ADAM 12 cysteine-rich domain-mediated cell adhesion, and then the beta1 integrin to induce cell spreading. Interestingly, carcinoma cells attached but did...
DEFF Research Database (Denmark)
Hansen-Schwartz, Jacob; Svensson, Carl-Lennart; Xu, Cang-Bao
2002-01-01
with ET-1 (unspecific ET(A) and ET(B) agonist), S6c (specific ET(B) agonist) and 5-CT (5-HT(1) agonist). Levels of mRNA coding for the ET(A), ET(B), 5-HT(1B) and 5-HT(1D) receptors were analysed using real-time RT-PCR. 3. Classical PKC's are critically involved in the appearance of the ET(B) receptor; co....... 2. The effect of inhibiting protein kinases during organ culture with staurosporine (unspecific protein kinase inhibitor), RO 31-7549 (specific inhibitor of classical PKC's) and H 89 (specific inhibitor of PKA) was examined using in vitro pharmacological examination of cultured vessel segments......-culture with RO 31-7549 abolished the contractile response (6.9 +/- 1.8%) and reduced the ET(B) receptor mRNA by 44 +/- 4% as compared to the cultured control. Correlation between decreased ET(B) receptor mRNA and abolished contractile function indicates upstream involvement of PKC. 4. Inhibition of PKA generally...
DEFF Research Database (Denmark)
Grüner, Sabine; Prostredna, Miroslava; Schulte, Valerie
2003-01-01
by intravital fluorescence microscopy that platelet adhesion and thrombus growth on the exposed ECM of the injured carotid artery is not significantly altered in alpha2-null mice and even in mice with a Cre/loxP-mediated loss of all beta1 integrins on their platelets. In contrast, inhibition of alphaIIbbeta3...... integrin on platelets in wild-type mice blocked aggregate formation and reduced platelet adhesion by 60.0%. Strikingly, alphaIIbbeta3 inhibition had a comparable effect in alpha2-null mice, demonstrating that other receptors mediate shear-resistant adhesion in the absence of functional alpha2beta1...... and alphaIIbbeta3. These were identified to be alpha5beta1 and/or alpha6beta1 as alphaIIbbeta3 inhibition abrogated platelet adhesion in beta1-null mice. We conclude that shear-resistant platelet adhesion on the injured vessel wall in vivo is a highly integrated process involving multiple integrin...
Ni, Xiuqin; Epshtein, Yulia; Chen, Weiguo; Zhou, Tingting; Xie, Lishi; Garcia, Joe G N; Jacobson, Jeffrey R
2014-06-01
We previously reported sphingosine 1-phosphate (S1P) and hepatocyte growth factor (HGF) augment endothelial cell (EC) barrier function and attenuate murine acute lung inury (ALI). While the mechanisms underlying these effects are not fully understood, S1P and HGF both transactivate the S1P receptor, S1PR1 and integrin β4 (ITGB4) at membrane caveolin-enriched microdomains (CEMs). In the current study, we investigated the roles of S1PR2 and S1PR3 in S1P/HGF-mediated EC signaling and their associations with ITGB4. Our studies confirmed ITGB4 and S1PR2/3 are recruited to CEMs in human lung EC in response to either S1P (1 µM, 5 min) or HGF (25 ng/ml, 5 min). Co-immunoprecipitation experiments identified an S1P/HGF-mediated interaction of ITGB4 with both S1PR2 and S1PR3. We then employed an in situ proximity ligation assay (PLA) to confirm a direct ITGB4-S1PR3 association induced by S1P/HGF although a direct association was not detectable between S1PR2 and ITGB4. S1PR1 knockdown (siRNA), however, abrogated S1P/HGF-induced ITGB4-S1PR2 associations while there was no effect on ITGB4-S1PR3 associations. Moreover, PLA confirmed a direct association between S1PR1 and S1PR2 induced by S1P and HGF. Finally, silencing of S1PR2 significantly attenuated S1P/HGF-induced EC barrier enhancement as measured by transendothelial resistance while silencing of S1PR3 significantly augmented S1P/HGF-induced barrier enhancement. These results confirm an important role for S1PR2 and S1PR3 in S1P/HGF-mediated EC barrier responses that are associated with their complex formation with ITGB4. Our findings elucidate novel mechanisms of EC barrier regulation that may ultimately lead to new therapeutic targets for disorders characterized by increased vascular permeability including ALI. © 2013 Wiley Periodicals, Inc.
Ren, Feng; Zhang, Hai-yan; Piao, Zheng-fu; Zheng, Su-jun; Chen, Yu; Chen, De-xi; Duan, Zhong-ping
2012-09-01
To determine the mechanism underlying the therapeutic activities of glycogen synthase kinase 3b (GSK3b) against hepatic ischemia-reperfusion (H-IR) injury by investigating the inhibitive effects of GSK3b on inflammation mediated by Toll-like receptor 4 (TLR4). C57BL/6 male mice were subjected to 90 min of warm liver cephalad lobe ischemia, followed by reperfusion for various lengths of time. The mice were divided into three groups: the H-IR untreated model (control group), and the H-IR inflammation-induced models that received an intraperitoneal injection of purified lipopolysaccharide (LPS) endotoxin alone (inflammation group) or with pretreatment of the SB216763 GSK3b-specific inhibitor (intervention group). To create a parallel isolated cell system for detailed investigations of macrophages, marrow-derived stem cells were isolated from femurs of the H-IR control group of mice and used to derive primary macrophages. The cells were then divided into the same three groups as the whole mouse system: control, LPS-induced inflammation model, and inflammation model with SB216763 intervention. Differential expressions of inflammation-related proteins and genes were detected by Western blotting and real-time quantitative PCR, respectively. The phosphorylation levels of ERK, JNK and p38 MAPK were induced in liver at 1 h after reperfusion, but then steadily decreased and returned to baseline levels by 4 h after reperfusion. In addition, the phosphorylation levels of ERK and JNK were induced in macrophages at 15 min after LPS stimulation, while the phosphorylation level of p38 MAPK was induced at 1 h; SB216763 pretreatment suppressed the LPS-stimulated ERK, JNK and p38 phosphorylation in macrophages. In the mouse model, GSK3b activity was found to promote the gene expression of anti-inflammatory cytokine IL-10 (control: 0.21 ± 0.08, inflammation: 0.83 ± 0.21, intervention: 1.76 ± 0.67; F = 3.16, P = 0.027) but to significantly inhibit the gene expression of pro
Navarro, Gemma; Borroto-Escuela, Dasiel; Angelats, Edgar; Etayo, Íñigo; Reyes-Resina, Irene; Pulido-Salgado, Marta; Rodríguez-Pérez, Ana I; Canela, Enric I; Saura, Josep; Lanciego, José Luis; Labandeira-García, José Luis; Saura, Carlos A; Fuxe, Kjell; Franco, Rafael
2018-01-01
Endocannabinoids are important regulators of neurotransmission and, acting on activated microglia, they are postulated as neuroprotective agents. Endocannabinoid action is mediated by CB 1 and CB 2 receptors, which may form heteromeric complexes (CB 1 -CB 2 Hets) with unknown function in microglia. We aimed at establishing the expression and signaling properties of cannabinoid receptors in resting and LPS/IFN-γ-activated microglia. In activated microglia mRNA transcripts increased (2 fold for CB 1 and circa 20 fold for CB 2 ), whereas receptor levels were similar for CB 1 and markedly upregulated for CB 2 ; CB 1 -CB 2 Hets were also upregulated. Unlike in resting cells, CB 2 receptors became robustly coupled to G i in activated cells, in which CB 1 -CB 2 Hets mediated a potentiation effect. Hence, resting cells were refractory while activated cells were highly responsive to cannabinoids. Interestingly, similar results were obtained in cultures treated with ß-amyloid (Aß 1-42 ). Microglial activation markers were detected in the striatum of a Parkinson's disease (PD) model and, remarkably, in primary microglia cultures from the hippocampus of mutant β-amyloid precursor protein (APP Sw,Ind ) mice, a transgenic Alzheimer's disease (AD) model. Also of note was the similar cannabinoid receptor signaling found in primary cultures of microglia from APP Sw,Ind and in cells from control animals activated using LPS plus IFN-γ. Expression of CB 1 -CB 2 Hets was increased in the striatum from rats rendered dyskinetic by chronic levodopa treatment. In summary, our results showed sensitivity of activated microglial cells to cannabinoids, increased CB 1 -CB 2 Het expression in activated microglia and in microglia from the hippocampus of an AD model, and a correlation between levodopa-induced dyskinesia and striatal microglial activation in a PD model. Cannabinoid receptors and the CB 1 -CB 2 heteroreceptor complex in activated microglia have potential as targets in the
Mahalingam, Bhuvaneshwari; Ajroud, Kaouther; Alonso, Jose Luis; Anand, Saurabh; Adair, Brian; Horenstein, Alberto L; Malavasi, Fabio; Xiong, Jian-Ping; Arnaout, M. Amin
2011-01-01
A central feature of integrin interaction with physiologic ligands is the monodentate binding of a ligand carboxylate to a Mg2+ ion hexacoordinated at the metal-ion-dependent-adhesion site (MIDAS) in the integrin A-domain. This interaction stabilizes the A-domain in the high-affinity state, which is distinguished from the default low-affinity state by tertiary changes in the domain that culminate in cell adhesion. Small molecule ligand-mimetic integrin antagonists act as partial agonists, eliciting similar activating conformational changes in the A-domain, which has contributed to paradoxical adhesion and increased patient mortality in large clinical trials. As with other ligand-mimetic integrin antagonists, the function-blocking monoclonal antibody (mAb) 107 binds MIDAS of integrin CD11b/CD18 A-domain (CD11bA), but in contrast, it favors the inhibitory Ca2+ ion over Mg2+ at MIDAS. We determined the crystal structures of the Fab fragment of mAb 107 complexed to the low- and high-affinity states of CD11bA. Favored binding of Ca2+ at MIDAS is caused by the unusual symmetric bidentate ligation of a Fab-derived ligand Asp to a heptacoordinated MIDAS Ca2+. Binding of Fab 107 to CD11bA did not trigger the activating tertiary changes in the domain or in the full-length integrin. These data show that denticity of the ligand Asp/Glu can modify divalent cation selectivity at MIDAS and hence integrin function. Stabilizing the Ca2+ ion at MIDAS by bidentate ligation to a ligand Asp/Glu may provide one approach for designing pure integrin antagonists. PMID:22095715
Lee, Hee Doo; Kim, Yeon Hyang; Kim, Doo-Sik
2014-04-01
Integrin trafficking, including internalization, recycling, and lysosomal degradation, is crucial for the regulation of cellular functions. Exosomes, nano-sized extracellular vesicles, are believed to play important roles in intercellular communications. This study demonstrates that exosomes released from human macrophages negatively regulate endothelial cell migration through control of integrin trafficking. Macrophage-derived exosomes promote internalization of integrin β1 in primary HUVECs. The internalized integrin β1 persistently accumulates in the perinuclear region and is not recycled back to the plasma membrane. Experimental results indicate that macrophage-derived exosomes stimulate trafficking of internalized integrin β1 to lysosomal compartments with a corresponding decrease in the integrin destined for recycling endosomes, resulting in proteolytic degradation of the integrin. Moreover, ubiquitination of HUVEC integrin β1 is enhanced by the exosomes, and exosome-mediated integrin degradation is blocked by bafilomycin A, a lysosomal degradation inhibitor. Macrophage-derived exosomes were also shown to effectively suppress collagen-induced activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway and HUVEC migration, which are both dependent on integrin β1. These observations provide new insight into the functional significance of exosomes in the regulation of integrin trafficking. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Directory of Open Access Journals (Sweden)
Beecken Wolf-Dietrich
2005-01-01
Full Text Available Abstract Background Tumor development remains one of the major obstacles following organ transplantation. Immunosuppressive drugs such as cyclosporine and tacrolimus directly contribute to enhanced malignancy, whereas the influence of the novel compound mycophenolate mofetil (MMF on tumor cell dissemination has not been explored. We therefore investigated the adhesion capacity of colon, pancreas, prostate and kidney carcinoma cell lines to endothelium, as well as their beta1 integrin expression profile before and after MMF treatment. Methods Tumor cell adhesion to endothelial cell monolayers was evaluated in the presence of 0.1 and 1 μM MMF and compared to unstimulated controls. beta1 integrin analysis included alpha1beta1 (CD49a, alpha2beta1 (CD49b, alpha3beta1 (CD49c, alpha4beta1 (CD49d, alpha5beta1 (CD49e, and alpha6beta1 (CD49f receptors, and was carried out by reverse transcriptase-polymerase chain reaction, confocal microscopy and flow cytometry. Results Adhesion of the colon carcinoma cell line HT-29 was strongly reduced in the presence of 0.1 μM MMF. This effect was accompanied by down-regulation of alpha3beta1 and alpha6beta1 surface expression and of alpha3beta1 and alpha6beta1 coding mRNA. Adhesion of the prostate tumor cell line DU-145 was blocked dose-dependently by MMF. In contrast to MMF's effects on HT-29 cells, MMF dose-dependently up-regulated alpha1beta1, alpha2beta1, alpha3beta1, and alpha5beta1 on DU-145 tumor cell membranes. Conclusion We conclude that MMF possesses distinct anti-tumoral properties, particularly in colon and prostate carcinoma cells. Adhesion blockage of HT-29 cells was due to the loss of alpha3beta1 and alpha6beta1 surface expression, which might contribute to a reduced invasive behaviour of this tumor entity. The enhancement of integrin beta1 subtypes observed in DU-145 cells possibly causes re-differentiation towards a low-invasive phenotype.
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Michael Andrew Meyer
2013-07-01
Full Text Available The JC polyoma viral coat protein VP1 was analyzed for amino acid sequences homologies to the IDSP sequence which mediates binding of VLA-4 (integrin alpha 4 to vascular cell adhesion molecule 1. Although the full sequence was not found, a DSP sequence was located near the critical arginine residue linked to infectivity of the virus and binding to sialic acid containing molecules such as integrins (3. For the JC polyoma virus, a DSP sequence was found at residues 70, 71 and 72 with homology also noted for the mouse polyoma virus and SV40 virus. Three dimensional modeling of the VP1 molecule suggests that the DSP loop has an accessible site for interaction from the external side of the assembled viral capsid pentamer.
Fibronectin-integrin signaling is required for L-glutamine's protection against gut injury.
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Stefanie Niederlechner
Full Text Available Extracellular matrix (ECM stabilization and fibronectin (FN-Integrin signaling can mediate cellular protection. L-glutamine (GLN is known to prevent apoptosis after injury. However, it is currently unknown if ECM stabilization and FN-Integrin osmosensing pathways are related to GLN's cell protective mechanism in the intestine.IEC-6 cells were treated with GLN with or without FN siRNA, integrin inhibitor GRGDSP, control peptide GRGESP or ERK1/2 inhibitors PD98059 and UO126 under basal and stressed conditions. Cell survival measured via MTS assay. Phosphorylated and/or total levels of cleaved caspase-3, cleaved PARP, Bax, Bcl-2, heat shock proteins (HSPs, ERK1/2 and transcription factor HSF-1 assessed via Western blotting. Cell size and F-actin morphology quantified by confocal fluorescence microscopy and intracellular GLN concentration by LC-MS/MS.GLN's prevention of FN degradation after hyperthermia attenuated apoptosis. Additionally, inhibition of FN-Integrin interaction by GRGDSP and ERK1/2 kinase inhibition by PD98059 inhibited GLN's protective effect. GRGDSP attenuated GLN-mediated increases in ERK1/2 phosphorylation and HSF-1 levels. PD98059 and GRGDSP also decreased HSP levels after GLN treatment. Finally, GRGDSP attenuated GLN-mediated increases in cell area size and disrupted F-actin assembly, but had no effect on intracellular GLN concentrations.Taken together, this data suggests that prevention of FN degradation and the FN-Integrin signaling play a key role in GLN-mediated cellular protection. GLN's signaling via the FN-Integrin pathway is associated with HSP induction via ERK1/2 and HSF-1 activation leading to reduced apoptosis after gut injury.
Integrin α6Bβ4 inhibits colon cancer cell proliferation and c-Myc activity
International Nuclear Information System (INIS)
Dydensborg, Anders Bondo; Teller, Inga C; Groulx, Jean-François; Basora, Nuria; Paré, Fréderic; Herring, Elizabeth; Gauthier, Rémy; Jean, Dominique; Beaulieu, Jean-François
2009-01-01
Integrins are known to be important contributors to cancer progression. We have previously shown that the integrin β4 subunit is up-regulated in primary colon cancer. Its partner, the integrin α6 subunit, exists as two different mRNA splice variants, α6A and α6B, that differ in their cytoplasmic domains but evidence for distinct biological functions of these α6 splice variants is still lacking. In this work, we first analyzed the expression of integrin α6A and α6B at the protein and transcript levels in normal human colonic cells as well as colorectal adenocarcinoma cells from both primary tumors and established cell lines. Then, using forced expression experiments, we investigated the effect of α6A and α6B on the regulation of cell proliferation in a colon cancer cell line. Using variant-specific antibodies, we observed that α6A and α6B are differentially expressed both within the normal adult colonic epithelium and between normal and diseased colonic tissues. Proliferative cells located in the lower half of the glands were found to predominantly express α6A, while the differentiated and quiescent colonocytes in the upper half of the glands and surface epithelium expressed α6B. A relative decrease of α6B expression was also identified in primary colon tumors and adenocarcinoma cell lines suggesting that the α6A/α6B ratios may be linked to the proliferative status of colonic cells. Additional studies in colon cancer cells showed that experimentally restoring the α6A/α6B balance in favor of α6B caused a decrease in cellular S-phase entry and repressed the activity of c-Myc. The findings that the α6Bβ4 integrin is expressed in quiescent normal colonic cells and is significantly down-regulated in colon cancer cells relative to its α6Aβ4 counterpart are consistent with the anti-proliferative influence and inhibitory effect on c-Myc activity identified for this α6Bβ4 integrin. Taken together, these findings point out the importance of integrin
Al-Rabia, Mohammed W; Blaylock, Morgan G; Sexton, Darren W; Walsh, Garry M
2004-06-01
Caspases are key molecules in the control of apoptosis, but relatively little is known about their contribution to eosinophil apoptosis. We examined caspase-3, -8, and -9 activities in receptor ligation-dependent apoptosis induction in the differentiated human eosinophilic cell line EoL-1. Differentiated EoL-1 exhibited bi-lobed nuclei, eosinophil-associated membrane receptors, and basic granule proteins. Annexin-V fluorescein isothiocyanate binding to EoL-1 revealed significant (PEoL-1 but had no effect on constitutive (baseline) apoptosis at 16 and 20 h. Caspase activity was analyzed using the novel CaspaTag trade mark technique and flow cytometry. EoL-1 treated with pan-CD45, CD45RA, CD45RB, and CD95 mAb exhibited caspase-3 and -9 activation at 12 h post-treatment, which increased at 16 and 20 h. Activated caspase-8 was detected 12 and 16 h after ligation with CD45, CD45RA, CD45RB, and CD95 mAb followed by a trend toward basal levels at 20 h. CD69 ligation resulted in caspase-3 activation, a modest but significant activation of caspase-8, and a loss in mitochondrial transmembrane potential but had no significant effect on activation of caspase-9. Thus, the intrinsic and extrinsic caspase pathways are involved in controlling receptor ligation-mediated apoptosis induction in human eosinophils, findings that may aid the development of a more targeted, anti-inflammatory therapy for asthma.
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Hozumi Kentaro
2016-11-01
Full Text Available Mimicking the biological function of the extracellular matrix is an approach to developing cell adhesive biomaterials. The RGD peptide, derived from fibronectin (Fn, mainly binds to integrin αvβ3 and has been widely used as a cell adhesive peptide on various biomaterials. However, cell adhesion to Fn is thought to be mediated by several integrin subtypes and syndecans. In this study, we synthesized an RGD-containing peptide (FIB1 and four integrin α4β1-binding-related motif-containing peptides (LDV, IDAPS, KLDAPT, and PRARI and constructed peptide-chitosan matrices. The FIB1-chitosan matrix promoted human dermal fibroblast (HDF attachment, and the C-terminal elongated PRARI (ePRARI-C-conjugated chitosan matrix significantly promoted HDF attachment through integrin α4β1 and syndecan binding. Next, we constructed a mixed ePRARI-C- and FIB1-chitosan matrix to develop a Fn mimetic biomaterial. The mixed ePRARI-C/FIB1-chitosan matrix promoted significantly better cell attachment and neurite outgrowth compared to those of either ePRARI-C- or FIB1-chitosan matrices. HDF adhesion to the ePRARI-C/FIB1-chitosan matrix was mediated by integrin, α4β1, α5β1, and αvβ3, similar to HDF adhesion to Fn. These data suggest that an ePRARI-C/FIB1-chitosan matrix can be used as a tool to analyze the multiple functions of Fn and can serve as a Fn-mimetic biomaterial.
International Nuclear Information System (INIS)
Slivka, S.R.; Insel, P.A.
1987-01-01
alpha 1-Adrenergic receptors mediate two effects on phospholipid metabolism in Madin-Darby canine kidney (MDCK-D1) cells: hydrolysis of phosphoinositides and arachidonic acid release with generation of prostaglandin E2 (PGE2). The similarity in concentration dependence for the agonist (-)-epinephrine in eliciting these two responses implies that they are mediated by a single population of alpha 1-adrenergic receptors. However, we find that the kinetics of the two responses are quite different, PGE2 production occurring more rapidly and transiently than the hydrolysis of phosphoinositides. The antibiotic neomycin selectively decreases alpha 1-receptor-mediated phosphatidylinositol 4,5-bisphosphate hydrolysis without decreasing alpha 1-receptor-mediated arachidonic acid release and PGE2 generation. In addition, receptor-mediated inositol trisphosphate formation is independent of extracellular calcium, whereas release of labeled arachidonic acid is largely calcium-dependent. Moreover, based on studies obtained with labeled arachidonic acid, receptor-mediated generation of arachidonic acid cannot be accounted for by breakdown of phosphatidylinositol monophosphate, phosphatidylinositol bisphosphate, or phosphatidic acid. Further studies indicate that epinephrine produces changes in formation or turnover of several classes of membrane phospholipids in MDCK cells. We conclude that alpha 1-adrenergic receptors in MDCK cells appear to regulate phospholipid metabolism by the parallel activation of phospholipase C and phospholipase A2. This parallel activation of phospholipases contrasts with models described in other systems which imply sequential activation of phospholipase C and diacylglycerol lipase or phospholipase A2
A complex relationship between TRAF3 and non-canonical NF-κB2 activation in B lymphocytes
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Wai Wai Lin
2013-12-01
Full Text Available The adaptor protein TRAF3 restrains BAFF receptor (BAFFR and CD40-mediated activation of the NF-κB2 pathway in B cells. Mice lacking TRAF3 specifically in B cells revealed the critical role of TRAF3 in restraining homeostatic B cell survival. Furthermore, loss- of-function mutations of the traf3 gene have been associated with human B cell malignancies, especially multiple myeloma (MM. It has been proposed that receptor-induced TRAF3 degradation leads to stabilization of the NF-B inducing kinase NIK, and subsequent NF-κB2 activation. However, it is unclear how receptor-mediated TRAF3 degradation or loss of function contributes to B cell-specific NF-κB2 activation. In the current study, we employed two complementary models to address this question. One utilized a mutant traf3 gene found in a human MM-derived cell line called LP1. The LP1 mutant TRAF3 protein lacks the TRAF-N and TRAF-C domains. Consistent with the paradigm described, expression of LP1 TRAF3 in B cells promoted higher basal levels of NF-κB2 activation compared to Wt TRAF3. However, LP1 did not associate with TRAF2, CD40, or BAFFR, and no LP1 degradation was observed following receptor engagement. Interestingly, LP1 showed enhanced NIK association. Thus, TRAF3 degradation becomes dispensable to activate NF-κB2 when it is unable to associate with TRAF2. In a second model, we examined several mutant forms of BAFFR that are unable to induce NF-κB2 activation in B cells. Signaling to B cells by each of these BAFFR mutants, however, induced levels of TRAF3 degradation similar to those induced by Wt BAFFR. Thus, in B cells, receptor-mediated TRAF3 degradation is not sufficient to promote NF-B2 activation. We thus conclude that there is not a simple linear relationship in B lymphocytes between relative levels of cellular TRAF3, induced TRAF3 degradation, NIK activation and NF-B2 activation.
Berrout, Liza; Isokawa, Masako
2018-01-01
Ghrelin and its receptor GHSR1a have been shown to exert numerous physiological functions in the brain, in addition to the well-established orexigenic role in the hypothalamus. Earlier work indicated that ghrelin stimulated the phosphorylation of the GluN1 subunit of the NMDA receptor (NMDAR) and enhanced synaptic transmission in the hippocampus. In the present study, we report that the exogenous application of ghrelin increased GluN2B phosphorylation. This increase was independent of GluN2B subunit activity or NMDAR channel activity. However, it depended on the activation of GHSR1a and Fyn as it was blocked by D-Lys3-GHRP-6 and PP2, respectively. Inhibitors for G-protein-regulated second messengers, such as Rp-cAMP, H89, TBB, ryanodine, and thapsigargin, unexpectedly enhanced GluN2B phosphorylation, suggesting that cAMP, PKA, casein kinase II, and cytosolic calcium signaling may oppose to the effect of ghrelin on the phosphorylation of GluN2B. Our findings suggest that 1) GluN2B is likely a molecular target of ghrelin and GHSR1a-driven signaling cascades, and 2) the ghrelin-mediated phosphorylation of GluN2B depends on Fyn activation under complex negative regulation by other second messengers. Copyright © 2017 Elsevier B.V. All rights reserved.
DEFF Research Database (Denmark)
Andreasen, Susanne Ø; Thomsen, Allan R; Koteliansky, Victor E
2003-01-01
decreased responses were seen upon transfer of alpha(1)-deficient activated/memory T cells. Thus, expression of alpha(1)beta(1) and alpha(2)beta(1) integrins on activated T cells is directly functionally important for generation of inflammatory responses within tissues. Finally, the inhibitory effect......Adhesive interactions are crucial to cell migration into inflammatory sites. Using murine lymphocytic choriomeningitis virus as an Ag model system, we have investigated expression and function of collagen-binding integrins, alpha(1)beta(1) and alpha(2)beta(1), on activated and memory T cells. Using...... this system and MHC tetramers to define Ag-specific T cells, we demonstrate that contrary to being VLAs, expression of alpha(1)beta(1) and alpha(2)beta(1) can be rapidly induced on acutely activated T cells, that expression of alpha(1)beta(1) remains elevated on memory T cells, and that expression of alpha(1...
Jia, Yuzhi; Viswakarma, Navin; Reddy, Janardan K
2014-01-01
Several nuclear receptors regulate diverse metabolic functions that impact on critical biological processes, such as development, differentiation, cellular regeneration, and neoplastic conversion. In the liver, some members of the nuclear receptor family, such as peroxisome proliferator-activated receptors (PPARs), constitutive androstane receptor (CAR), farnesoid X receptor (FXR), liver X receptor (LXR), pregnane X receptor (PXR), glucocorticoid receptor (GR), and others, regulate energy homeostasis, the formation and excretion of bile acids, and detoxification of xenobiotics. Excess energy burning resulting from increases in fatty acid oxidation systems in liver generates reactive oxygen species, and the resulting oxidative damage influences liver regeneration and liver tumor development. These nuclear receptors are important sensors of exogenous activators as well as receptor-specific endogenous ligands. In this regard, gene knockout mouse models revealed that some lipid-metabolizing enzymes generate PPARα-activating ligands, while others such as ACOX1 (fatty acyl-CoA oxidase1) inactivate these endogenous PPARα activators. In the absence of ACOX1, the unmetabolized ACOX1 substrates cause sustained activation of PPARα, and the resulting increase in energy burning leads to hepatocarcinogenesis. Ligand-activated nuclear receptors recruit the multisubunit Mediator complex for RNA polymerase II-dependent gene transcription. Evidence indicates that the Med1 subunit of the Mediator is essential for PPARα, PPARγ, CAR, and GR signaling in liver. Med1 null hepatocytes fail to respond to PPARα activators in that these cells do not show induction of peroxisome proliferation and increases in fatty acid oxidation enzymes. Med1-deficient hepatocytes show no increase in cell proliferation and do not give rise to liver tumors. Identification of nuclear receptor-specific coactivators and Mediator subunits should further our understanding of the complexities of metabolic
Saranya, Jayaram; Shilpa, Ganesan; Raghu, Kozhiparambil G.; Priya, Sulochana
2017-01-01
Lectins are a unique class of carbohydrate binding proteins/glycoproteins, and many of them possess anticancer properties. They can induce cell cycle arrest and apoptosis, inhibit protein synthesis, telomerase activity and angiogenesis in cancer cells. In the present study, we have demonstrated the effect of Morus alba leaf lectin (MLL) on anoikis induction in MCF-7 cells. Anoikis induction in cancer cells has a significant role in preventing early stage metastasis. MLL treatment in monolayers of MCF-7 cells caused significant detachment of cells in a time and concentration dependent manner. The detached cells failed to re-adhere and grew even to culture plates coated with different matrix proteins. DNA fragmentation, membrane integrity studies, annexin V staining, caspase 9 activation and upregulation of Bax/Bad confirmed that the detached cells underwent apoptosis. Upregulation of matrix metalloproteinase 9 (MMP-9) caused a decrease in fibronectin (FN) production which facilitated the cells to detach by blocking the FN mediated downstream signaling. On treatment with MLL, we have observed downregulation of integrin expression, decreased phosphorylation of focal adhesion kinase (FAK), loss in FAK-integrin interaction and active Ras. MLL treatment downregulated the levels of phosphorylated Akt and PI3K. Also, we have studied the effect of MLL on two stress activated protein kinases p38 MAPK and JNK. p38 MAPK activation was found to be elevated, but there was no change in the level of JNK. Thus our study substantiated the possible antimetastatic effect of MLL by inducing anoikis in MCF-7 cells by activation of caspase 9 and proapoptotic Bax/Bad by blockage of FN mediated integrin/FAK signaling and partly by activation of p38 MAPK. PMID:28223935
Sphingosine 1-phosphate receptor activation enhances BMP-2-induced osteoblast differentiation
International Nuclear Information System (INIS)
Sato, Chieri; Iwasaki, Tsuyoshi; Kitano, Sachie; Tsunemi, Sachi; Sano, Hajime
2012-01-01
Highlights: ► We investigated the role of S1P signaling for osteoblast differentiation. ► Both S1P and FTY enhanced BMP-2-stimulated osteoblast differentiation by C2C12 cells. ► S1P signaling enhanced BMP-2-stimulated Smad and ERK phosphorylation by C2C12 cells. ► MEK/ERK signaling is a pathway underlying S1P signaling for osteoblast differentiation. -- Abstract: We previously demonstrated that sphingosine 1-phosphate (S1P) receptor-mediated signaling induced proliferation and prostaglandin productions by synovial cells from rheumatoid arthritis (RA) patients. In the present study we investigated the role of S1P receptor-mediated signaling for osteoblast differentiation. We investigated osteoblast differentiation using C2C12 myoblasts, a cell line derived from murine satellite cells. Osteoblast differentiation was induced by the treatment of bone morphogenic protein (BMP)-2 in the presence or absence of either S1P or FTY720 (FTY), a high-affinity agonist of S1P receptors. Osteoblast differentiation was determined by osteoblast-specific transcription factor, Runx2 mRNA expression, alkaline phosphatase (ALP) activity and osteocalcin production by the cells. Smad1/5/8 and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation was examined by Western blotting. Osteocalcin production by C2C12 cells were determined by ELISA. Runx2 expression and ALP activity by BMP-2-stimulated C2C12 cells were enhanced by addition of either S1P or FTY. Both S1P and FTY enhanced BMP-2-induced ERK1/2 and Smad1/5/8 phosphorylation. The effect of FTY was stronger than that of S1P. S1P receptor-mediated signaling on osteoblast differentiation was inhibited by addition of mitogen-activated protein kinase/ERK kinase (MEK) 1/2 inhibitor, indicating that the S1P receptor-mediated MEK1/2-ERK1/2 signaling pathway enhanced BMP-2-Smad signaling. These results indicate that S1P receptor-mediated signaling plays a crucial role for osteoblast differentiation.
LFA-1 and Mac-1 integrins bind to the serine/threonine-rich domain of thrombomodulin
Energy Technology Data Exchange (ETDEWEB)
Kawamoto, Eiji [Department of Molecular Pathobiology and Cell Adhesion Biology, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507 (Japan); Emergency and Critical Care Center, Mie University Hospital, 2-174 Edobashi, Tsu 514-8507 (Japan); Okamoto, Takayuki, E-mail: okamotot@doc.medic.mie-u.ac.jp [Department of Molecular Pathobiology and Cell Adhesion Biology, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507 (Japan); Takagi, Yoshimi [Department of Molecular Pathobiology and Cell Adhesion Biology, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507 (Japan); Honda, Goichi [Medical Affairs Department, Asahi Kasei Pharma Corporation, 1-105 Kanda Jinbo-cho, Chiyoda-ku, Tokyo 101-8101 (Japan); Suzuki, Koji [Faculty of Pharmaceutical Science, Suzuka University of Medical Science, 3500-3, Minamitamagaki-cho, Suzuka, Mie 513-8679 (Japan); Imai, Hiroshi [Emergency and Critical Care Center, Mie University Hospital, 2-174 Edobashi, Tsu 514-8507 (Japan); Shimaoka, Motomu, E-mail: shimaoka@doc.medic.mie-u.ac.jp [Department of Molecular Pathobiology and Cell Adhesion Biology, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507 (Japan)
2016-05-13
LFA-1 (αLβ2) and Mac-1 (αMβ2) integrins regulate leukocyte trafficking in health and disease by binding primarily to IgSF ligand ICAM-1 and ICAM-2 on endothelial cells. Here we have shown that the anti-coagulant molecule thrombomodulin (TM), found on the surface of endothelial cells, functions as a potentially new ligand for leukocyte integrins. We generated a recombinant extracellular domain of human TM and Fc fusion protein (TM-domains 123-Fc), and showed that pheripheral blood mononuclear cells (PBMCs) bind to TM-domains 123-Fc dependent upon integrin activation. We then demonstrated that αL integrin-blocking mAb, αM integrin-blocking mAb, and β2 integrin-blocking mAb inhibited the binding of PBMCs to TM-domains 123-Fc. Furthermore, we show that the serine/threonine-rich domain (domain 3) of TM is required for the interaction with the LFA-1 (αLβ2) and Mac-1 (αMβ2) integrins to occur on PBMCs. These results demonstrate that the LFA-1 and Mac-1 integrins on leukocytes bind to TM, thereby establishing the molecular and structural basis underlying LFA-1 and Mac-1 integrin interaction with TM on endothelial cells. In fact, integrin-TM interactions might be involved in the dynamic regulation of leukocyte adhesion with endothelial cells. - Highlights: • LFA-1 and Mac-1 integrins bind to the anti-coagulant molecule thrombomodulin. • The serine/threonine-rich domain of thrombomodulin is essential to interact with the LFA-1 and Mac-1 integrins on PBMCs. • Integrin-TM interactions might be involved in the dynamic regulation of leukocyte adhesion with endothelial cells.
Nicotine impairs cyclooxygenase-2-dependent kinin-receptor-mediated murine airway relaxations
International Nuclear Information System (INIS)
Xu, Yuan; Cardell, Lars-Olaf
2014-01-01
Introduction: Cigarette smoke induces local inflammation and airway hyperreactivity. In asthmatics, it worsens the symptoms and increases the risk for exacerbation. The present study investigates the effects of nicotine on airway relaxations in isolated murine tracheal segments. Methods: Segments were cultured for 24 h in the presence of vehicle, nicotine (10 μM) and/or dexamethasone (1 μM). Airway relaxations were assessed in myographs after pre-contraction with carbachol (1 μM). Kinin receptors, cyclooxygenase (COX) and inflammatory mediator expressions were assessed by real-time PCR and confocal-microscopy-based immunohistochemistry. Results: The organ culture procedure markedly increased bradykinin- (selective B 2 receptor agonist) and des-Arg 9 -bradykinin- (selective B 1 receptor agonist) induced relaxations, and slightly increased relaxation induced by isoprenaline, but not that induced by PGE 2 . The kinin receptor mediated relaxations were epithelium-, COX-2- and EP2-receptor-dependent and accompanied by drastically enhanced mRNA levels of kinin receptors, as well as inflammatory mediators MCP-1 and iNOS. Increase in COX-2 and mPGES-1 was verified both at mRNA and protein levels. Nicotine selectively suppressed the organ-culture-enhanced relaxations induced by des-Arg 9 -bradykinin and bradykinin, at the same time reducing mPGES-1 mRNA and protein expressions. α7-nicotinic acetylcholine receptor inhibitors α-bungarotoxin and MG624 both blocked the nicotine effects on kinin B 2 receptors, but not those on B 1 . Dexamethasone completely abolished kinin-induced relaxations. Conclusion: It is tempting to conclude that a local inflammatory process per se could have a bronchoprotective component by increasing COX-2 mediated airway relaxations and that nicotine could impede this safety mechanism. Dexamethasone further reduced airway inflammation together with relaxations. This might contribute to the steroid resistance seen in some patients with asthma
Nicotine impairs cyclooxygenase-2-dependent kinin-receptor-mediated murine airway relaxations
Energy Technology Data Exchange (ETDEWEB)
Xu, Yuan, E-mail: yuan.xu@ki.se; Cardell, Lars-Olaf
2014-02-15
Introduction: Cigarette smoke induces local inflammation and airway hyperreactivity. In asthmatics, it worsens the symptoms and increases the risk for exacerbation. The present study investigates the effects of nicotine on airway relaxations in isolated murine tracheal segments. Methods: Segments were cultured for 24 h in the presence of vehicle, nicotine (10 μM) and/or dexamethasone (1 μM). Airway relaxations were assessed in myographs after pre-contraction with carbachol (1 μM). Kinin receptors, cyclooxygenase (COX) and inflammatory mediator expressions were assessed by real-time PCR and confocal-microscopy-based immunohistochemistry. Results: The organ culture procedure markedly increased bradykinin- (selective B{sub 2} receptor agonist) and des-Arg{sup 9}-bradykinin- (selective B{sub 1} receptor agonist) induced relaxations, and slightly increased relaxation induced by isoprenaline, but not that induced by PGE{sub 2}. The kinin receptor mediated relaxations were epithelium-, COX-2- and EP2-receptor-dependent and accompanied by drastically enhanced mRNA levels of kinin receptors, as well as inflammatory mediators MCP-1 and iNOS. Increase in COX-2 and mPGES-1 was verified both at mRNA and protein levels. Nicotine selectively suppressed the organ-culture-enhanced relaxations induced by des-Arg{sup 9}-bradykinin and bradykinin, at the same time reducing mPGES-1 mRNA and protein expressions. α7-nicotinic acetylcholine receptor inhibitors α-bungarotoxin and MG624 both blocked the nicotine effects on kinin B{sub 2} receptors, but not those on B{sub 1}. Dexamethasone completely abolished kinin-induced relaxations. Conclusion: It is tempting to conclude that a local inflammatory process per se could have a bronchoprotective component by increasing COX-2 mediated airway relaxations and that nicotine could impede this safety mechanism. Dexamethasone further reduced airway inflammation together with relaxations. This might contribute to the steroid resistance seen in
Expression Profile of the Integrin Receptor Subunits in the Guinea Pig Sclera.
Wang, Kevin K; Metlapally, Ravikanth; Wildsoet, Christine F
2017-06-01
The ocular dimensional changes in myopia reflect increased scleral remodeling, and in high myopia, loss of scleral integrity leads to biomechanical weakening and continued scleral creep. As integrins, a type of cell surface receptors, have been linked to scleral remodeling, they represent potential targets for myopia therapies. As a first step, this study aimed to characterize the integrin subunits at the messenger RNA level in the sclera of the guinea pig, a more recently added but increasingly used animal model for myopia research. Primers for α and β integrin subunits were designed using NCBI/UCSC Genome Browser and Primer3 software tools. Total RNA was extracted from normal scleral tissue and isolated cultured scleral fibroblasts, as well as liver and lung, as reference tissues, all from guinea pig. cDNA was produced by reverse transcription, PCR was used to amplify products of predetermined sizes, and products were sequenced using standard methods. Guinea pig scleral tissue expressed all known integrin alpha subunits except αD and αE. The latter integrin subunits were also not expressed by cultured guinea pig scleral fibroblasts; however, their expression was confirmed in guinea pig liver. In addition, isolated cultured fibroblasts did not express integrin subunits αL, αM, and αX. This difference between results for cultured cells and intact sclera presumably reflects the presence in the latter of additional cell types. Both guinea pig scleral tissue and isolated scleral fibroblasts expressed all known integrin beta subunits. All results were verified through sequencing. The possible contributions of integrins to scleral remodeling make them plausible targets for myopia prevention. Data from this study will help guide future ex vivo and in vitro studies directed at understanding the relationship between scleral integrins and ocular growth regulation in the guinea pig model for myopia.
The CD157-Integrin Partnership Controls Transendothelial Migration and Adhesion of Human Monocytes*
Lo Buono, Nicola; Parrotta, Rossella; Morone, Simona; Bovino, Paola; Nacci, Giulia; Ortolan, Erika; Horenstein, Alberto L.; Inzhutova, Alona; Ferrero, Enza; Funaro, Ada
2011-01-01
CD157, a member of the CD38 gene family, is an NAD-metabolizing ectoenzyme and a signaling molecule whose role in polarization, migration, and diapedesis of human granulocytes has been documented; however, the molecular events underpinning this role remain to be elucidated. This study focused on the role exerted by CD157 in monocyte migration across the endothelial lining and adhesion to extracellular matrix proteins. The results demonstrated that anti-CD157 antibodies block monocyte transmigration and adhesion to fibronectin and fibrinogen but that CD157 cross-linking is sufficient to overcome the block, suggesting an active signaling role for the molecule. Consistent with this is the observation that CD157 is prevalently located within the detergent-resistant membrane microdomains to which, upon clustering, it promotes the recruitment of β1 and β2 integrin, which, in turn, leads to the formation of a multimolecular complex favoring signal transduction. This functional cross-talk with integrins allows CD157 to act as a receptor despite its intrinsic structural inability to do so on its own. Intracellular signals mediated by CD157 rely on the integrin/Src/FAK (focal adhesion kinase) pathway, resulting in increased activity of the MAPK/ERK1/2 and the PI3K/Akt downstream signaling pathways, which are crucial in the control of monocyte transendothelial migration. Collectively, these findings indicate that CD157 acts as a molecular organizer of signaling-competent membrane microdomains and that it forms part of a larger molecular machine ruled by integrins. The CD157-integrin partnership provides optimal adhesion and transmigration of human monocytes. PMID:21478153
Mezzasoma, Letizia; Antognelli, Cinzia; Talesa, Vincenzo Nicola
2016-02-01
Atrial natriuretic peptide (ANP) is an hormone/paracrine/autocrine factor regulating cardiovascular homeostasis by guanylyl cyclase natriuretic peptide receptor (NPR-1). ANP plays an important role also in regulating inflammatory and immune systems by altering macrophages functions and cytokines secretion. Interleukin-1β (IL-1β) is a potent pro-inflammatory cytokine involved in a wide range of biological responses, including the immunological one. Unlike other cytokines, IL-1β production is rigorously controlled. Primarily, NF-kB activation is required to produce pro-IL-1β; subsequently, NALP3 inflammasome/caspase-1 activation is required to cleave pro-IL-1β into the active secreted protein. NALP3 is a molecular platform capable of sensing a large variety of signals and a major player in innate immune defense. Due to their pleiotropism, IL-1β and NALP3 dysregulation is a common feature of a wide range of diseases. Therefore, identifying molecules regulating IL-1β/NALP3/caspase-1 expression is an important step in the development of new potential therapeutic agents. The aim of our study was to evaluate the effect of ANP on IL-1β/NALP3/caspase-1 expression in LPS/ATP-stimulated human THP1 monocytes. We provided new evidence of the direct involvement of ANP/NPR-1/cGMP axis on NF-kB/NALP3/caspase-1-mediated IL-1β release and NF-kB-mediated pro-IL-1β production. In particular, ANP inhibited both NF-kB and NALP3/caspase-1 activation leading to pro- and mature IL-1β down-regulation. Our data, pointing out a modulatory role of this endogenous peptide on IL-1β release and on NF-kB/NALP3/caspase-1 activation, indicate an important anti-inflammatory and immunomodulatory effect of ANP via these mechanisms. We suggest a possible employment of ANP for the treatment of inflammatory/immune-related diseases and IL-1β/NALP3-associated disorders, affecting millions of people worldwide.
Wong, Mei Mei; Chen, Yikuan; Margariti, Andriani; Winkler, Bernhard; Campagnolo, Paola; Potter, Claire; Hu, Yanhua; Xu, Qingbo
2014-03-01
Vascular lineage differentiation of stem/progenitor cells can contribute to both tissue repair and exacerbation of vascular diseases such as in vein grafts. The role of macrophages in controlling vascular progenitor differentiation is largely unknown and may play an important role in graft development. This study aims to identify the role of macrophages in vascular stem/progenitor cell differentiation and thereafter elucidate the mechanisms that are involved in the macrophage- mediated process. We provide in vitro evidence that macrophages can induce endothelial cell (EC) differentiation of the stem/progenitor cells while simultaneously inhibiting their smooth muscle cell differentiation. Mechanistically, both effects were mediated by macrophage-derived tumor necrosis factor-α (TNF-α) via TNF-α receptor 1 and canonical nuclear factor-κB activation. Although the overexpression of p65 enhanced EC (or attenuated smooth muscle cell) differentiation, p65 or TNF-α receptor 1 knockdown using lentiviral short hairpin RNA inhibited EC (or rescued smooth muscle cell) differentiation in response to TNF-α. Furthermore, TNF-α-mediated EC differentiation was driven by direct binding of nuclear factor-κB (p65) to specific VE-cadherin promoter sequences. Subsequent experiments using an ex vivo decellularized vessel scaffold confirmed an increase in the number of ECs and reduction in smooth muscle cell marker expression in the presence of TNF-α. The lack of TNF-α in a knockout mouse model of vein graft decreased endothelialization and significantly increased thrombosis formation. Our study highlights the role of macrophages in directing vascular stem/progenitor cell lineage commitment through TNF-α-mediated TNF-α receptor 1 and nuclear factor-κB activation that is likely required for endothelial repair in vascular diseases such as vein graft.
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Wu Jian
2009-11-01
Full Text Available Abstract Background Phosphatase of regenerating liver-3 (PRL-3 plays a causative role in tumor metastasis, but the underlying mechanisms are not well understood. In our previous study, we observed that PRL-3 could decrease tyrosine phosphorylation of integrin β1 and enhance activation of ERK1/2 in HEK293 cells. Herein we aim to explore the association of PRL-3 with integrin β1 signaling and its functional implications in motility, invasion, and metastasis of colon cancer cell LoVo. Methods Transwell chamber assay and nude mouse model were used to study motility and invasion, and metastsis of LoVo colon cancer cells, respectively. Knockdown of integrin β1 by siRNA or lentivirus were detected with Western blot and RT-PCR. The effect of PRL-3 on integrin β1, ERK1/2, and MMPs that mediate motility, invasion, and metastasis were measured by Western blot, immunofluorencence, co-immunoprecipitation and zymographic assays. Results We demonstrated that PRL-3 associated with integrin β1 and its expression was positively correlated with ERK1/2 phosphorylation in colon cancer tissues. Depletion of integrin β1 with siRNA, not only abrogated the activation of ERK1/2 stimulated by PRL-3, but also abolished PRL-3-induced motility and invasion of LoVo cells in vitro. Similarly, inhibition of ERK1/2 phosphorylation with U0126 or MMP activity with GM6001 also impaired PRL-3-induced invasion. In addition, PRL-3 promoted gelatinolytic activity of MMP2, and this stimulation correlated with decreased TIMP2 expression. Moreover, PRL-3-stimulated lung metastasis of LoVo cells in a nude mouse model was inhibited when integrin β1 expression was interfered with shRNA. Conclusion Our results suggest that PRL-3's roles in motility, invasion, and metastasis in colon cancer are critically controlled by the integrin β1-ERK1/2-MMP2 signaling.
Discoidin Domain Receptor 1 Mediates Myosin-Dependent Collagen Contraction
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Nuno M. Coelho
2017-02-01
Full Text Available Discoidin domain receptor 1 (DDR1 is a tyrosine kinase collagen adhesion receptor that mediates cell migration through association with non-muscle myosin IIA (NMIIA. Because DDR1 is implicated in cancer fibrosis, we hypothesized that DDR1 interacts with NMIIA to enable collagen compaction by traction forces. Mechanical splinting of rat dermal wounds increased DDR1 expression and collagen alignment. In periodontal ligament of DDR1 knockout mice, collagen mechanical reorganization was reduced >30%. Similarly, cultured cells with DDR1 knockdown or expressing kinase-deficient DDR1d showed 50% reduction of aligned collagen. Tractional remodeling of collagen was dependent on DDR1 clustering, activation, and interaction of the DDR1 C-terminal kinase domain with NMIIA filaments. Collagen remodeling by traction forces, DDR1 tyrosine phosphorylation, and myosin light chain phosphorylation were increased on stiff versus soft substrates. Thus, DDR1 clustering, activation, and interaction with NMIIA filaments enhance the collagen tractional remodeling that is important for collagen compaction in fibrosis.
Purinergic Receptors: Key Mediators of HIV-1 infection and inflammation
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Talia H Swartz
2015-11-01
Full Text Available Human immunodeficiency virus (HIV-1 causes a chronic infection that afflicts more than 38 million individuals worldwide. While the infection can be suppressed with potent anti-retroviral therapies, individuals infected with HIV have elevated levels of inflammation as indicated by increased T cell activation, soluble biomarkers, and associated morbidity and mortality. A single mechanism linking HIV pathogenesis to this inflammation has yet to be identified. Purinergic receptors are known to mediate inflammation and have been shown to be required for HIV-1 infection at the level of HIV-1 membrane fusion. Here we review the literature on the role of purinergic receptors in HIV-1 infection and associated inflammation and describe a role for these receptors as potential therapeutic targets.
Directory of Open Access Journals (Sweden)
Rong Jin
Full Text Available Recent work has revealed an essential involvement of soluble CD40L (sCD40L in inflammation and vascular disease. Activated platelets are the major source of sCD40L, which has been implicated in platelet and leukocyte activation, although its exact functional impact on leukocyte-platelet interactions and the underlying mechanisms remain undefined. We aimed to determine the impact and the mechanisms of sCD40L on neutrophils. We studied neutrophil interactions with activated, surface-adherent platelets as a model for leukocyte recruitment to the sites of injury. Our data show that CD40L contributes to neutrophil firm adhesion to and transmigration across activated surface-adherent platelets, possibly through two potential mechanisms. One involves the direct interaction of ligand-receptor (CD40L-CD40, i.e., platelet surface CD40L interaction with neutrophil CD40; another involves an indirect mechanism, i.e. soluble CD40L stimulates activation of the leukocyte-specific β2 integrin Mac-1 in neutrophils and thereby further promotes neutrophil adhesion and migration. Activation of the integrin Mac-1 is known to be critical for mediating neutrophil adhesion and migration. sCD40L activated Mac-1 in neutrophils and enhanced neutrophil-platelet interactions in wild-type neutrophils, but failed to elicit such responses in CD40-deficient neutrophils. Furthermore, our data show that the protein kinase C zeta (PKCζ is critically required for sCD40L-induced Mac-1 activation and neutrophil adhesive function. sCD40L strongly stimulated the focal clustering of Mac-1 (CD11b and the colocalization of Mac-1 with PKCζ in wild-type neutrophils, but had minimal effect in CD40-deficient neutrophils. Blocking PKCζ completely inhibited sCD40L-induced neutrophil firm adhesion. Moreover, sCD40L strongly stimulates neutrophil oxidative burst via CD40-dependent activation of PI3K/NF-KB, but independent of Mac-1 and PKCζ. These findings may contribute to a better
Directory of Open Access Journals (Sweden)
Yang Qi
2009-12-01
Full Text Available Abstract The midbrain periaqueductal grey (PAG is a structure known for its roles in pain transmission and modulation. Noxious stimuli potentiate the glutamate synaptic transmission and enhance glutamate NMDA receptor expression in the PAG. However, little is known about roles of NMDA receptor subunits in the PAG in processing the persistent inflammatory pain. The present study was undertaken to investigate NR2A- and NR2B-containing NMDA receptors in the PAG and their modulation to the peripheral painful inflammation. Noxious stimuli induced by hind-paw injection of complete Freund's adjuvant (CFA caused up-regulation of NR2B-containing NMDA receptors in the PAG, while NR2A-containing NMDA receptors were not altered. Whole-cell patch-clamp recordings revealed that NMDA receptor mediated mEPSCs were increased significantly in the PAG synapse during the chronic phases of inflammatory pain in mice. PAG local infusion of Ro 25-6981, an NR2B antagonist, notably prolonged the paw withdrawal latency to thermal radian heat stimuli bilaterally in rats. Hyperoside (Hyp, one of the flavonoids compound isolated from Rhododendron ponticum L., significantly reversed up-regulation of NR2B-containing NMDA receptors in the PAG and exhibited analgesic activities against persistent inflammatory stimuli in mice. Our findings provide strong evidence that up-regulation of NR2B-containing NMDA receptors in the PAG involves in the modulation to the peripheral persistent inflammatory pain.
The role of scavenger receptor B1 in infection with Mycobacterium tuberculosis in a murine model.
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Georgia Schäfer
2009-12-01
Full Text Available The interaction between Mycobacterium tuberculosis (Mtb and host cells is complex and far from being understood. The role of the different receptor(s implicated in the recognition of Mtb in particular remains poorly defined, and those that have been found to have activity in vitro were subsequently shown to be redundant in vivo.To identify novel receptors involved in the recognition of Mtb, we screened a macrophage cDNA library and identified scavenger receptor B class 1 (SR-B1 as a receptor for mycobacteria. SR-B1 has been well-described as a lipoprotein receptor which mediates both the selective uptake of cholesteryl esters and the efflux of cholesterol, and has also recently been implicated in the recognition of other pathogens. We show here that mycobacteria can bind directly to SR-B1 on transfected cells, and that this interaction could be inhibited in the presence of a specific antibody to SR-B1, serum or LDL. We define a variety of macrophage populations, including alveolar macrophages, that express this receptor, however, no differences in the recognition and response to mycobacteria were observed in macrophages isolated from SR-B1(-/- or wild type mice in vitro. Moreover, when wild type and SR-B1(-/- animals were infected with a low dose of Mtb (100 CFU/mouse there were no alterations in survival, bacterial burdens, granuloma formation or cytokine production in the lung. However, significant reduction in the production of TNF, IFNgamma, and IL10 were observed in SR-B1(-/- mice following infection with a high dose of Mtb (1000 CFU/mouse, which marginally affected the size of inflammatory foci but did not influence bacterial burdens. Deficiency of SR-B1 also had no effect on resistance to disease under conditions of varying dietary cholesterol. We did observe, however, that the presence of high levels of cholesterol in the diet significantly enhanced the bacterial burdens in the lung, but this was independent of SR-B1.SR-B1 is involved in
Halder, Sebok K; Kant, Ravi; Milner, Richard
2018-05-01
Spinal cord injury (SCI) leads to rapid destruction of neuronal tissue, resulting in devastating motor and sensory deficits. This is exacerbated by damage to spinal cord blood vessels and loss of vascular integrity. Thus, approaches that protect existing blood vessels or stimulate the growth of new blood vessels might present a novel approach to minimize loss or promote regeneration of spinal cord tissue following SCI. In light of the remarkable power of chronic mild hypoxia (CMH) to stimulate vascular remodeling in the brain, the goal of this study was to examine how CMH (8% O 2 for up to 7 days) affects blood vessel remodeling in the spinal cord. We found that CMH promoted the following: (1) endothelial proliferation and increased vascularity as a result of angiogenesis and arteriogenesis, (2) increased vascular expression of the angiogenic extracellular matrix protein fibronectin as well as concomitant increases in endothelial expression of the fibronectin receptor α5β1 integrin, (3) strongly upregulated endothelial expression of the tight junction proteins claudin-5, ZO-1 and occludin and (4) astrocyte activation. Of note, the vascular remodeling changes induced by CMH were more extensive in white matter. Interestingly, hypoxic-induced vascular remodeling in spinal cord blood vessels was markedly attenuated in mice lacking endothelial α5 integrin expression (α5-EC-KO mice). Taken together, these studies demonstrate the considerable remodeling potential of spinal cord blood vessels and highlight an important angiogenic role for the α5β1 integrin in promoting endothelial proliferation. They also imply that stimulation of the α5β1 integrin or controlled use of mild hypoxia might provide new approaches for promoting angiogenesis and improving vascular integrity in spinal cord blood vessels.
Rawlings, N G; Simko, E; Bebchuk, T; Caldwell, S J; Singh, B
2003-07-01
Melanomas are common neoplasms of dogs and arise from pigment-producing cells called melanocytes or melanoblasts. Melanomas of skin are often easily cured by surgical excision, but those of oral mucosa are aggressive, metastasize to the regional lymph nodes and lungs, and respond poorly to conventional therapy. Tumor growth is sustained by proliferation of microvessels via a process called angiogenesis. Integrin alpha(v)beta3 is expressed in proliferating but not in quiescent microvessels suggesting a role in angiogenesis. Vascular endothelial growth factor (VEGF) manifests its mitogenic and angiogenic effects mainly via VEGF receptor-2 (VEGFR-2/Flk-1). We conducted this immunocytochemical study to investigate the expression of integrin alpha(v)beta3 and VEGFR-2 in archival and fresh samples from cases of canine melanomas. Results show that integrin alpha(v)beta3 was expressed in 72% and 88% of cutaneous and oral melanomas, respectively, and the expression was restricted to and immediately around the melanocytes and endothelial cells. VEGFR-2 staining of selected cases of melanoma revealed that its expression overlapped with the alpha(v)beta3 integrin. Dual immuno-gold electron microscopy confirmed co-localization of integrin alpha(v)beta3 and VEGFR-2 in melanocytes and endothelial cells. These data demonstrate expression and co-localization of integrin alpha(v)beta3 and VEGFR-2 in cutaneous and oral melanomas of dogs.
The metabolic activator FOXO1 binds hepatitis B virus DNA and activates its transcription
International Nuclear Information System (INIS)
Shlomai, Amir; Shaul, Yosef
2009-01-01
Hepatitis B virus (HBV) is a small DNA virus that targets the liver and infects humans worldwide. Recently we have shown that the metabolic regulator PGC-1α coactivates HBV transcription thereby rendering the virus susceptible to fluctuations in the nutritional status of the liver. PGC-1α coactivation of HBV is mediated through the liver-enriched nuclear receptor HNF4α and through another yet unknown transcription factor(s). Here we show that the forkhead transcription factor FOXO1, a known target for PGC-1α coactivation and a central mediator of glucose metabolism in the liver, binds HBV core promoter and activates its transcription. This activation is further enhanced in the presence of PGC-1α, implying that FOXO1 is a target for PGC-1α coactivation of HBV transcription. Thus, our results identify another key metabolic regulator as an activator of HBV transcription, thereby supporting the principle that HBV gene expression is regulated in a similar way to key hepatic metabolic genes.
Schwarz, Lindsay A; Hall, Benjamin J; Patrick, Gentry N
2010-12-08
The accurate trafficking of AMPA receptors (AMPARs) to and from the synapse is a critical component of learning and memory in the brain, whereas dysfunction of AMPAR trafficking is hypothesized to be an underlying mechanism of Alzheimer's disease. Previous work has shown that ubiquitination of integral membrane proteins is a common posttranslational modification used to mediate endocytosis and endocytic sorting of surface proteins in eukaryotic cells. Here we report that mammalian AMPARs become ubiquitinated in response to their activation. Using a mutant of GluA1 that is unable to be ubiquitinated at lysines on its C-terminus, we demonstrate that ubiquitination is required for internalization of surface AMPARs and their trafficking to the lysosome in response to the AMPAR agonist AMPA but not for internalization of AMPARs in response to the NMDA receptor agonist NMDA. Through overexpression or RNA interference-mediated knockdown, we identify that a specific E3 ligase, Nedd4-1 (neural-precursor cell-expressed developmentally downregulated gene 4-1), is necessary for this process. Finally, we show that ubiquitination of GluA1 by Nedd4-1 becomes more prevalent as neurons mature. Together, these data show that ubiquitination of GluA1-containing AMPARs by Nedd4-1 mediates their endocytosis and trafficking to the lysosome. Furthermore, these results provide insight into how hippocampal neurons regulate AMPAR trafficking and degradation with high specificity in response to differing neuronal signaling cues and suggest that changes to this pathway may occur as neurons mature.
Energy Technology Data Exchange (ETDEWEB)
Gabriel, L.M.; Soares, M.A.; Bicalho, M.S.; Santos, R.G. [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil)], e-mail: marcellaaraugio@yahoo.com.br, e-mail: mbs@cdtn.br, e-mail: santosr@cdtn.br; Sanchez, E.O.F.; Silva, S.G. [Ezequiel Dias Foundation, Belo Horizonte, MG (Brazil)], e-mail: silea@funed.mg.gov.br, e-mail: eladio@funed.mg.gov.br
2009-07-01
Integrins are family of heterodimeric cell surface adhesion receptors able to recognize and bind to proteins in the extracellular matrix (ECM). This recognition is mainly through the RGD domain present in both the cell surface as the protein in the ECM. Various integrins have been identified as regulators of tumor progression. The RGD domain is also found in some snake venoms named disintegrins. Disintegrins inhibit cell-matrix and a cell-cell interaction mediated by integrin and has been shown that these proteins are able to inhibit metastasis in processes dependent on integrin. The disintegrin-like (ECD), as well as RGD-disintegrin are also able to bind to cell surface integrins and inhibit their adherence to the natural ligands. Leucurolysin-B (Leuc-B) is a metalloproteinase class P-III isolated from Bothrops leucurus (BLV) and possesses a disintegrin-like domain (ECD). The goals of this work were to synthesize a radioactive probe analog to Leuc-B using radioiodine {sup 125}I and evaluate the interaction of {sup 125}I-Leuc-B in tumor cells through the study of biodistribution in animals bearing Ehrlich tumor.125I-Leuc-B was synthesized using lactoperoxidase with high yield (90%) and specific activity of 1.2x10-7Bq/mmol. It was observed that {sup 125}I-Leuc-B had very fast clearance from the blood stream (T1/2= 0.01 h). Tumor uptake of 125I-Leuc-B gradually increased up to (2 min) and remained for a quite long period. The tumor/normal tissue uptake ratios of {sup 125}I-Leuc-B were 1.77 (tumor/normal paw) and 8.44 tumor/skeletal muscle. The results suggest that {sup 125}I-Leuc- B may constitute a good template for development of a tool for detection of solid tumors. (author)
International Nuclear Information System (INIS)
Gabriel, L.M.; Soares, M.A.; Bicalho, M.S.; Santos, R.G.; Sanchez, E.O.F.; Silva, S.G.
2009-01-01
Integrins are family of heterodimeric cell surface adhesion receptors able to recognize and bind to proteins in the extracellular matrix (ECM). This recognition is mainly through the RGD domain present in both the cell surface as the protein in the ECM. Various integrins have been identified as regulators of tumor progression. The RGD domain is also found in some snake venoms named disintegrins. Disintegrins inhibit cell-matrix and a cell-cell interaction mediated by integrin and has been shown that these proteins are able to inhibit metastasis in processes dependent on integrin. The disintegrin-like (ECD), as well as RGD-disintegrin are also able to bind to cell surface integrins and inhibit their adherence to the natural ligands. Leucurolysin-B (Leuc-B) is a metalloproteinase class P-III isolated from Bothrops leucurus (BLV) and possesses a disintegrin-like domain (ECD). The goals of this work were to synthesize a radioactive probe analog to Leuc-B using radioiodine 125 I and evaluate the interaction of 125 I-Leuc-B in tumor cells through the study of biodistribution in animals bearing Ehrlich tumor.125I-Leuc-B was synthesized using lactoperoxidase with high yield (90%) and specific activity of 1.2x10-7Bq/mmol. It was observed that 125 I-Leuc-B had very fast clearance from the blood stream (T1/2= 0.01 h). Tumor uptake of 125I-Leuc-B gradually increased up to (2 min) and remained for a quite long period. The tumor/normal tissue uptake ratios of 125 I-Leuc-B were 1.77 (tumor/normal paw) and 8.44 tumor/skeletal muscle. The results suggest that 125 I-Leuc- B may constitute a good template for development of a tool for detection of solid tumors. (author)
International Nuclear Information System (INIS)
Landau-Levin, Mary; Chao, Clifford K.S.
1996-01-01
membrane receptors were used. Immunoperoxidase staining was performed. The resulting integrin expression was evaluated by two observers in a blinded fashion. The consensus score (CS) was calculated by the following equation: SC = Σ Pi (i + 1), where i = 1, 2, 3 (indicating weak, moderate, or strong staining) and Pi is the percentage of stained epithelial cells for each intensity, varying from 0 to 100%. Multiple logistic regression was performed to evaluate the association between integrin expression and invasive/metastatic propensity in these four groups. Associations were obtained using Mantel-Hazel correlation and P values were calculated by Fisher's exact test. Results and Conclusions: We found the expression of αv domain was significantly associated with the presence of LNM (P<0.01). Expression of β4 domain decreased in those tumors invading outer third cervical stroma (P<0.02). The correlation between the integrin expression adjusting for other known confounding prognosticators in predicting the aggressiveness of human cervical cancer and the potential clinical application in selecting tumors suitable for radiation therapy will be discussed
Larsen, Michele Campaigne; Brake, Paul B; Pollenz, Richard S; Jefcoate, Colin R
2004-11-01
Cytochrome P4501B1 (CYP1B1), the major constitutively expressed CYP in the rat mammary gland, is induced by Ah-receptor (AhR) ligands, while CYP1A1 is predominantly expressed only after induction. These CYPs contribute to carcinogenic activation of polycyclic aromatic hydrocarbons (PAHs). AhR, ARNT, and CYP1B1 were only weakly expressed, even after 2,3,7,8-tetrachlorodibenzo-p-dioxin induction, when rat mammary epithelial cells (RMEC) were cultured on plastic. RMEC cultured on the extracellular matrix (ECM), Matrigel, or on a floating gel of collagen I demonstrated branching morphogenesis and substantially increased basal CYP1B1 and induced CYP1A1 expression, in parallel with large increases in AhR and ARNT expression. Branching was more pronounced in the Wistar Kyoto than in the Wistar Furth rat strain. Although EGF enhanced branching, neither strain nor growth factor treatment substantially impacted CYP expression. Increased AhR and ARNT expression is observed within 24 h of dispersal on Matrigel, substantially prior to branch formation. Culture on thin layers of collagen I, collagen IV, and laminin, respectively, failed to reproduce the branching morphogenesis or increases in AhR, ARNT, or CYP expression. However, adherent, gelled collagen I recapitulated the increased protein expression, without supporting branching. This increased protein expression was closely paralleled by enhanced expression of beta-catenin and E-cadherin, components of cell-cell adhesion complexes. A synthetic peptide that selectively antagonizes integrin-ECM interactions reduced branch formation, without diminishing AhR, ARNT, and CYP expression. These data demonstrate that early ECM surface adhesion interactions mediate AhR and ARNT expression, which enhances CYP expression, independent of branching morphogenesis.
Zhu, Jieqing; Choi, Won-Seok; McCoy, Joshua G; Negri, Ana; Zhu, Jianghai; Naini, Sarasija; Li, Jihong; Shen, Min; Huang, Wenwei; Bougie, Daniel; Rasmussen, Mark; Aster, Richard; Thomas, Craig J; Filizola, Marta; Springer, Timothy A; Coller, Barry S
2012-03-14
An integrin found on platelets, α(IIb)β(3) mediates platelet aggregation, and α(IIb)β(3) antagonists are effective antithrombotic agents in the clinic. Ligands bind to integrins in part by coordinating a magnesium ion (Mg(2+)) located in the β subunit metal ion-dependent adhesion site (MIDAS). Drugs patterned on the integrin ligand sequence Arg-Gly-Asp have a basic moiety that binds the α(IIb) subunit and a carboxyl group that coordinates the MIDAS Mg(2+) in the β(3) subunits. They induce conformational changes in the β(3) subunit that may have negative consequences such as exposing previously hidden epitopes and inducing the active conformation of the receptor. We recently reported an inhibitor of α(IIb)β(3) (RUC-1) that binds exclusively to the α(IIb) subunit; here, we report the structure-based design and synthesis of RUC-2, a RUC-1 derivative with a ~100-fold higher affinity. RUC-2 does not induce major conformational changes in β(3) as judged by monoclonal antibody binding, light scattering, gel chromatography, electron microscopy, and a receptor priming assay. X-ray crystallography of the RUC-2-α(IIb)β(3) headpiece complex in 1 mM calcium ion (Ca(2+))/5 mM Mg(2+) at 2.6 Å revealed that RUC-2 binds to α(IIb) the way RUC-1 does, but in addition, it binds to the β(3) MIDAS residue glutamic acid 220, thus displacing Mg(2+) from the MIDAS. When the Mg(2+) concentration was increased to 20 mM, however, Mg(2+) was identified in the MIDAS and RUC-2 was absent. RUC-2's ability to inhibit ligand binding and platelet aggregation was diminished by increasing the Mg(2+) concentration. Thus, RUC-2 inhibits ligand binding by a mechanism different from that of all other α(IIb)β(3) antagonists and may offer advantages as a therapeutic agent.
Melanocortin MC(4) receptor-mediated feeding and grooming in rodents.
Mul, Joram D; Spruijt, Berry M; Brakkee, Jan H; Adan, Roger A H
2013-11-05
Decades ago it was recognized that the pharmacological profile of melanocortin ligands that stimulated grooming behavior in rats was strikingly similar to that of Xenopus laevis melanophore pigment dispersion. After cloning of the melanocortin MC1 receptor, expressed in melanocytes, and the melanocortin MC4 receptor, expressed mainly in brain, the pharmacological profiles of these receptors appeared to be very similar and it was demonstrated that these receptors mediate melanocortin-induced pigmentation and grooming respectively. Grooming is a low priority behavior that is concerned with care of body surface. Activation of central melanocortin MC4 receptors is also associated with meal termination, and continued postprandial stimulation of melanocortin MC4 receptors may stimulate natural postprandial grooming behavior as part of the behavioral satiety sequence. Indeed, melanocortins fail to suppress food intake or induce grooming behavior in melanocortin MC4 receptor-deficient rats. This review will focus on how melanocortins affect grooming behavior through the melanocortin MC4 receptor, and how melanocortin MC4 receptors mediate feeding behavior. This review also illustrates how melanocortins were the most likely candidates to mediate grooming and feeding based on the natural behaviors they induced. Copyright © 2013 Elsevier B.V. All rights reserved.
International Nuclear Information System (INIS)
Cordes, N.; Beinke, C.; Beuningen, D. van; Plasswilm, L.
2004-01-01
Background and purpose: interactions of cells with a substratum, especially extracellular matrix proteins, initiate clustering of integrin receptors in the cell membrane. This process represents the initial step for the activation of signaling pathways regulating survival, proliferation, differentiation, adhesion, and migration, and could, furthermore, be important for cellular resistance-mediating mechanisms against radiation or cytotoxic drugs. The lack of data elucidating the impact of irradiation or cytotoxic drugs on this important phenomenon led to this study on human A549 lung cancer cells in vitro. Material and methods: the human lung carcinoma cell line A549 grown on polystyrene or fibronectin (FN) was irradiated with 0-8 Gy or treated with cisplatin (0.1-50 μM), paclitaxel (0.1-50 nM), or mitomycin (0.1-50 μM). Colony formation assays, immunofluorescence staining in combination with activation of integrin clustering using anti-β 1 -integrin antibodies (K20), and Western blotting for tyrosine phosphorylation under treatment of cells with the IC 50 for irradiation (2 Gy; IC 50 = 2.2 Gy), cisplatin (2 μM), paclitaxel (5 nM), or mitomycin (7 μM) were performed. Results: attachment of cells to FN resulted in a significantly reduced radio- and chemosensitivity compared to polystyrene. The clustering of β 1 -integrins examined by immunofluorescence staining was only stimulated by irradiation, cisplatin, paclitaxel, or mitomycin in case of cell attachment to FN. By contrast, tyrosine phosphorylation, as one of the major events following β 1 -integrin clustering, showed a 3.7-fold, FN-related enhancement, and treatment of cells with the IC 50 of radiation, cisplatin, paclitaxel, or mitomycin showed a substratum-dependent induction. Conclusion: for the first time, a strong influence of irradiation and a variety of cytotoxic drugs on the clustering of β 1 -integrins could be shown. This event is a prerequisite for tyrosine phosphorylation and, thus, the
Marcinkiewicz, Cezary; Gerstenhaber, Jonathan A; Sternberg, Mark; Lelkes, Peter I; Feuerstein, Giora
2017-01-01
Thromboembolic events (TEE) underwrite key causes of death in developed countries. While advanced imaging technologies such as computed tomography scans serve to diagnose blood clots during acute cardiovascular events, no such technology is available in routine primary care for TEE risk assessment. Here, we describe an imaging platform technology based on bioengineered fluorescent nanodiamond particles (F-NDPs) functionalized with bitistatin (Bit), a disintegrin that specifically binds to the α IIb β 3 integrin, platelet fibrinogen receptor (PFR) on activated platelets. Covalent linkage of purified Bit to F-NDP was concentration-dependent and saturable, as validated by enzyme-linked immunosorbent assay using specific anti-Bit antibodies. F-NDP-Bit interacted with purified PFR, either in immobilized or soluble form. Lotrafiban, a nonpeptide, α IIb β 3 receptor antagonist, specifically blocked F-NDP-Bit-PFR complex formation. Moreover, F-NDP-Bit specifically binds to activated platelets incorporated into a clot generated by thrombin-activated rat platelet-rich plasma (PRP). Our results suggest that engineered F-NDP-Bit particles could serve as noninvasive, "real-time" optical diagnostics for clots present in blood vessels.
International Nuclear Information System (INIS)
Sandhu, Hardip; Xu, Cang Bao; Edvinsson, Lars
2010-01-01
Cigarette smoke exposure increases the risk of stroke. However, the underlying molecular mechanisms are poorly understood. Endothelin system plays key roles in the pathogenesis of stroke. The present study was designed to examine if lipid-soluble (dimethyl sulfoxide-soluble) cigarette smoke particles (DSP) induces upregulation of contractile endothelin type B (ET B ) receptors in rat cerebral arteries and if activation of mitogen activated protein kinase (MAPK) and nuclear factor-kappaB (NF-κB) mediate the upregulation of contractile endothelin receptors in the cerebral arteries. Rat middle cerebral arteries were isolated and organ cultured in serum free medium for 24 h in the presence of DSP with or without specific inhibitors: MEK specific (U0126), p38 specific (SB202190), JNK specific (SP600125), NF-κB specific (BMS-345541) or (IMD-0354), transcription inhibitor (actinomycin D), or translation blocker (cycloheximide). Contractile responses to the ET B receptor agonist sarafotoxin 6c were investigated by a sensitive myograph. The expression of the ET B receptors were studied at mRNA and protein levels using quantitative real time PCR and immunohistochemistry, respectively. Results show that organ culture per se induced transcriptional upregulation of contractile ET B receptors in the cerebral vascular smooth muscle cells. This upregulation was further increased at the translational level by addition of DSP to the organ culture, but this increase was not seen by addition of nicotine or water-soluble cigarette smoke particles to the organ culture. The increased upregulation of contractile ET B receptors by DSP was abrogated by U0126, SP600125, actinomycin D, and cycloheximide, suggesting that the underlying molecular mechanisms involved in this process include activation of MEK and JNK MAPK-mediated transcription and translation of new contractile ET B receptors. Thus, the MAPK-mediated upregulation of contractile ET B receptors in cerebral arteries might be a
Krebs, Kristi; Ruusmann, Anu; Simonlatser, Grethel; Velling, Teet
2015-12-01
FLNa is a ubiquitous cytoskeletal protein that links transmembrane receptors, including integrins, to F-actin and functions as a signalling intermediate. We investigated FLNa's role in the function of integrin-type collagen receptors, EGF-EGFR signalling and regulation of PKB/Akt and ERK1/2. Using FLNa-deficient M2 human melanoma cells, and same cells expressing EGFP-FLNa (M2F) or its Ig-like repeats 1-8+24, 8-15+24 and 16-24, we found that in M2F and M2 8-15+24 cells, EGF induced the increased phosphorylation of PKB/Akt and ERK1/2. In M2F cells EGF induced the localisation of these kinases to cell nucleus and lamellipodia, respectively, and the ERK1/2 phosphorylation-dependent co-immunoprecipitation of FLNa with ERK1/2. Only M2F and M2 8-15+24 cells adhered to and spread on type I collagen whereas on fibronectin all cells behaved similarly. α1β1 and α2β1 were the integrin-type collagen receptors expressed on these cells with primarily α1β1 localising to focal contacts and affecting cell adhesion and migration in a manner dependent on FLNa or its Ig-like repeats 8-15. Our results suggest a role for FLNa repeats 8-15 in the α1-subunit-dependent regulation of integrin α1β1 function, EGF-EGFR signalling to PKB/Akt and ERK1/2, identify ERK1/2 in EGF-induced FLNa-associated protein complexes, and show that the function of different integrins is subjected to differential regulation by FLNa. Copyright © 2015. Published by Elsevier GmbH.
Activation states of blood eosinophils in asthma
Johansson, Mats W.
2014-01-01
Asthma is characterized by airway inflammation rich in eosinophils. Airway eosinophilia is associated with exacerbations and has been suggested to play a role in airway remodeling. Recruitment of eosinophils from the circulation requires that blood eosinophils become activated, leading to their arrest on the endothelium and extravasation. Circulating eosinophils can be envisioned as potentially being in different activation states, including non-activated, pre-activated or “primed”, or fully activated. In addition, the circulation can potentially be deficient of pre-activated or activated eosinophils, because such cells have marginated on activated endothelium or extravasated into the tissue. A number of eosinophil-surface proteins, including CD69, L-selectin, intercellular adhesion molecule-1 (ICAM-1, CD54), CD44, P-selectin glycoprotein ligand-1 (PSGL-1, CD162), cytokine receptors, Fc receptors, integrins including αM integrin (CD11b), and activated conformations of Fc receptors and integrins have been proposed to report cell activation. Variation in eosinophil activation states may be associated with asthma activity. Eosinophil-surface proteins proposed to be activation markers, with a particular focus on integrins, and evidence for associations between activation states of blood eosinophils and features of asthma are reviewed here. Partial activation of β1 and β2 integrins on blood eosinophils, reported by monoclonal antibodies (mAb) N29 and KIM-127, is associated with impaired pulmonary function and airway eosinophilia, respectively, in non-severe asthma. The association with lung function does not occur in severe asthma, presumably due to greater eosinophil extravasation, specifically of activated or pre-activated cells, in severe disease. PMID:24552191
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Frank Scott Hall
Full Text Available Knockout (KO mice that lack the dopamine transporter (SL6A3; DAT display increased locomotion that can be attenuated, under some circumstances, by administration of drugs that normally produce psychostimulant-like effects, such as amphetamine and methylphenidate. These results have led to suggestions that DAT KO mice may model features of attention deficit hyperactivity disorder (ADHD and that these drugs may act upon serotonin (5-HT systems to produce these unusual locomotor decreasing effects. Evidence from patterns of brain expression and initial pharmacologic studies led us to use genetic and pharmacologic approaches to examine the influence of altered 5-HT1B receptor activity on hyperactivity in DAT KO mice. Heterozygous 5-HT1B KO and pharmacologic 5-HT1B antagonism both attenuated locomotor hyperactivity in DAT KO mice. Furthermore, DAT KO mice with reduced, but not eliminated, 5-HT1B receptor expression regained cocaine-stimulated locomotion, which was absent in DAT KO mice with normal levels of 5-HT1B receptor expression. Further experiments demonstrated that the degree of habituation to the testing apparatus determined whether cocaine had no effect on locomotion in DAT KO or reduced locomotion, helping to resolve differences among prior reports. These findings of complementation of the locomotor effects of DAT KO by reducing 5-HT1B receptor activity underscore roles for interactions between specific 5-HT receptors and dopamine (DA systems in basal and cocaine-stimulated locomotion and support evaluation of 5-HT1B antagonists as potential, non-stimulant ADHD therapeutics.
Liu, Peihong; Du, Yongli; Song, Lianhua; Shen, Jingkang; Li, Qunyi
2016-08-08
Protein tyrosine phosphatase 1B (PTP1B) as a key negative regulator of both insulin and leptin receptor pathways has been an attractive therapeutic target for the treatment of type 2 diabetes mellitus (T2DM) and obesity. With the goal of enhancing potency and selectivity of the PTP1B inhibitors, a series of methyl salicylate derivatives as ABC type PTP1B inhibitors (P1-P7) were discovered. More importantly, compound P6 exhibited high potent inhibitory activity (IC50 = 50 nM) for PTP1B with 15-fold selectivity over T-cell PTPase (TCPTP). Further studies on cellular activities revealed that compound P6 could enhance insulin-mediated insulin receptor β (IRβ) phosphorylation and insulin-stimulated glucose uptake. Copyright © 2016 Elsevier Masson SAS. All rights reserved.
Lee, Jin Ju; Simborio, Hannah Leah; Reyes, Alisha Wehdnesday Bernardo; Hop, Huynh Tan; Arayan, Lauren Togonon; Lee, Hu Jang; Min, Wongi; Her, Moon; Rhee, Man Hee; Watarai, Masahisa; Chang, Hong Hee; Kim, Suk
2016-04-21
Brucella abortus is an intracellular pathogen which can infect and persist in host cells through multiple interactions. Above all, its interaction to host cell receptor is important to understand the pathogenic mechanisms of B. abortus. Accordingly, we demonstrated that platelet-activating factor receptor (PAFR) affects host cell response against B. abortus infection. First of all, B. abortus infection to macrophage induces secretion of platelet-activating factor (PAF), which is a PAFR agonist. The stimulation of PAFR by PAF remarkably increases B. abortus uptake into macrophages. It induces Janus kinase 2 (JAK2) and p38α phosphorylation, indicating that PAFR-mediated activation of JAK2 signaling leads to enhanced uptake of B. abortus. Moreover, the dynamics of F-actin polymerization revealed that PAFR-mediated B. abortus uptake is related with the reorganization of F-actin and JAK2. Upon B. abortus phagocytosis, reduced PAFR in the membrane and subsequently increased levels of PAFR colocalization with endosomes were observed which indicate that B. abortus uptake into macrophages allowed PAFR trafficking to endosomes. This study demonstrated that PAFR has a compelling involvement in B. abortus uptake as a promoter of phagocytosis, which is associated with JAK2 activation. Thus, our findings establish a novel insight into a receptor-related phagocytic mechanism of B. abortus.
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Mei-Fei Yueh
Full Text Available Triclocarban (3,4,4'-trichlorocarbanilide, TCC is used as a broad-based antimicrobial agent that is commonly added to personal hygiene products. Because of its extensive use in the health care industry and resistance to degradation in sewage treatment processes, TCC has become a significant waste product that is found in numerous environmental compartments where humans and wildlife can be exposed. While TCC has been linked to a range of health and environmental effects, few studies have been conducted linking exposure to TCC and induction of xenobiotic metabolism through regulation by environmental sensors such as the nuclear xenobiotic receptors (XenoRs. To identify the ability of TCC to activate xenobiotic sensors, we monitored XenoR activities in response to TCC treatment using luciferase-based reporter assays. Among the XenoRs in the reporter screening assay, TCC promotes both constitutive androstane receptor (CAR and estrogen receptor alpha (ERα activities. TCC treatment to hUGT1 mice resulted in induction of the UGT1A genes in liver. This induction was dependent upon the constitutive active/androstane receptor (CAR because no induction occurred in hUGT1Car(-/- mice. Induction of the UGT1A genes by TCC corresponded with induction of Cyp2b10, another CAR target gene. TCC was demonstrated to be a phenobarbital-like activator of CAR in receptor-based assays. While it has been suggested that TCC be classified as an endocrine disruptor, it activates ERα leading to induction of Cyp1b1 in female ovaries as well as in promoter activity. Activation of ERα by TCC in receptor-based assays also promotes induction of human CYP2B6. These observations demonstrate that TCC activates nuclear xenobiotic receptors CAR and ERα both in vivo and in vitro and might have the potential to alter normal physiological homeostasis. Activation of these xenobiotic-sensing receptors amplifies gene expression profiles that might represent a mechanistic base for
Sphingosine 1-phosphate receptor activation enhances BMP-2-induced osteoblast differentiation
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Sato, Chieri [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan); Iwasaki, Tsuyoshi, E-mail: tsuyo-i@huhs.ac.jp [Division of Pharmacotherapy, Department of Pharmacy, School of Pharmacy, Hyogo University of Health Sciences, 1-3-6 Minatojima, Chuo-ku, Kobe 650-8530 (Japan); Kitano, Sachie; Tsunemi, Sachi; Sano, Hajime [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan)
2012-06-22
Highlights: Black-Right-Pointing-Pointer We investigated the role of S1P signaling for osteoblast differentiation. Black-Right-Pointing-Pointer Both S1P and FTY enhanced BMP-2-stimulated osteoblast differentiation by C2C12 cells. Black-Right-Pointing-Pointer S1P signaling enhanced BMP-2-stimulated Smad and ERK phosphorylation by C2C12 cells. Black-Right-Pointing-Pointer MEK/ERK signaling is a pathway underlying S1P signaling for osteoblast differentiation. -- Abstract: We previously demonstrated that sphingosine 1-phosphate (S1P) receptor-mediated signaling induced proliferation and prostaglandin productions by synovial cells from rheumatoid arthritis (RA) patients. In the present study we investigated the role of S1P receptor-mediated signaling for osteoblast differentiation. We investigated osteoblast differentiation using C2C12 myoblasts, a cell line derived from murine satellite cells. Osteoblast differentiation was induced by the treatment of bone morphogenic protein (BMP)-2 in the presence or absence of either S1P or FTY720 (FTY), a high-affinity agonist of S1P receptors. Osteoblast differentiation was determined by osteoblast-specific transcription factor, Runx2 mRNA expression, alkaline phosphatase (ALP) activity and osteocalcin production by the cells. Smad1/5/8 and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation was examined by Western blotting. Osteocalcin production by C2C12 cells were determined by ELISA. Runx2 expression and ALP activity by BMP-2-stimulated C2C12 cells were enhanced by addition of either S1P or FTY. Both S1P and FTY enhanced BMP-2-induced ERK1/2 and Smad1/5/8 phosphorylation. The effect of FTY was stronger than that of S1P. S1P receptor-mediated signaling on osteoblast differentiation was inhibited by addition of mitogen-activated protein kinase/ERK kinase (MEK) 1/2 inhibitor, indicating that the S1P receptor-mediated MEK1/2-ERK1/2 signaling pathway enhanced BMP-2-Smad signaling. These results indicate that S1P
Directory of Open Access Journals (Sweden)
Stefanie Löffek
Full Text Available The hemidesmosomal transmembrane component collagen XVII (ColXVII plays an important role in the anchorage of the epidermis to the underlying basement membrane. However, this adhesion protein seems to be also involved in the regulation of keratinocyte migration, since its expression in these cells is strongly elevated during reepithelialization of acute wounds and in the invasive front of squamous cell carcinoma, while its absence in ColXVII-deficient keratinocytes leads to altered cell motility. Using a genetic model of murine Col17a1⁻/⁻ keratinocytes we elucidated ColXVII mediated signaling pathways in cell adhesion and migration. Col17a1⁻/⁻ keratinocytes exhibited increased spreading on laminin 332 and accelerated, but less directed cell motility. These effects were accompanied by increased expression of the integrin subunits β4 and β1. The migratory phenotype, as evidenced by formation of multiple unstable lamellipodia, was associated with enhanced phosphoinositide 3-kinase (PI3K activity. Dissection of the signaling pathway uncovered enhanced phosphorylation of the β4 integrin subunit and the focal adhesion kinase (FAK as activators of PI3K. This resulted in elevated Rac1 activity as a downstream consequence. These results provide mechanistic evidence that ColXVII coordinates keratinocyte adhesion and directed motility by interfering integrin dependent PI3K activation and by stabilizing lamellipodia at the leading edge of reepithelializing wounds and in invasive squamous cell carcinoma.
Protease-activated receptor-2 activation exaggerates TRPV1-mediated cough in guinea pigs.
Gatti, Raffaele; Andre, Eunice; Amadesi, Silvia; Dinh, Thai Q; Fischer, Axel; Bunnett, Nigel W; Harrison, Selena; Geppetti, Pierangelo; Trevisani, Marcello
2006-08-01
A lowered threshold to the cough response frequently accompanies chronic airway inflammatory conditions. However, the mechanism(s) that from chronic inflammation results in a lowered cough threshold is poorly understood. Irritant agents, including capsaicin, resiniferatoxin, and citric acid, elicit cough in humans and in experimental animals through the activation of the transient receptor potential vanilloid 1 (TRPV1). Protease-activated receptor-2 (PAR2) activation plays a role in inflammation and sensitizes TRPV1 in cultured sensory neurons by a PKC-dependent pathway. Here, we have investigated whether PAR2 activation exaggerates TRPV1-dependent cough in guinea pigs and whether protein kinases are involved in the PAR2-induced cough modulation. Aerosolized PAR2 agonists (PAR2-activating peptide and trypsin) did not produce any cough per se. However, they potentiated citric acid- and resiniferatoxin-induced cough, an effect that was completely prevented by the TRPV1 receptor antagonist capsazepine. In contrast, cough induced by hypertonic saline, a stimulus that provokes cough in a TRPV1-independent manner, was not modified by aerosolized PAR2 agonists. The PKC inhibitor GF-109203X, the PKA inhibitor H-89, and the cyclooxygenase inhibitor indomethacin did not affect cough induced by TRPV1 agonists, but abated the exaggeration of this response produced by PAR2 agonists. In conclusion, PAR2 stimulation exaggerates TRPV1-dependent cough by activation of diverse mechanism(s), including PKC, PKA, and prostanoid release. PAR2 activation, by sensitizing TRPV1 in primary sensory neurons, may play a role in the exaggerated cough observed in certain airways inflammatory diseases such as asthma and chronic obstructive pulmonary disease.
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Wang, Yanling; Fälting, Johanna M.; Mattsson, Charlotte L.; Holmström, Therése E.; Nedergaard, Jan, E-mail: jan@metabol.su.se
2013-10-15
Brown adipose tissue is unusual in that the neurotransmitter norepinephrine influences cell destiny in ways generally associated with effects of classical growth factors: regulation of cell proliferation, of apoptosis, and progression of differentiation. The norepinephrine effects are mediated through G-protein-coupled receptors; further mediation of such stimulation to e.g. Erk1/2 activation is in cell biology in general accepted to occur through transactivation of the EGF receptor (by external or internal pathways). We have examined here the significance of such transactivation in brown adipocytes. Stimulation of mature brown adipocytes with cirazoline (α{sub 1}-adrenoceptor coupled via G{sub q}), clonidine (α{sub 2} via G{sub i}) or CL316243 (β{sub 3} via G{sub s}) or via β{sub 1}-receptors significantly activated Erk1/2. Pretreatment with the EGF receptor kinase inhibitor AG1478 had, remarkably, no significant effect on Erk1/2 activation induced by any of these adrenergic agonists (although it fully abolished EGF-induced Erk1/2 activation), demonstrating absence of EGF receptor-mediated transactivation. Results with brown preadipocytes (cells in more proliferative states) were not qualitatively different. Joint stimulation of all adrenoceptors with norepinephrine did not result in synergism on Erk1/2 activation. AG1478 action on EGF-stimulated Erk1/2 phosphorylation showed a sharp concentration–response relationship (IC{sub 50} 0.3 µM); a minor apparent effect of AG1478 on norepinephrine-stimulated Erk1/2 phosphorylation showed nonspecific kinetics, implying caution in interpretation of partial effects of AG1478 as reported in other systems. Transactivation of the EGF receptor is clearly not a universal prerequisite for coupling of G-protein coupled receptors to Erk1/2 signalling cascades. - Highlights: • In brown adipocytes, norepinephrine regulates proliferation, apoptosis, differentiation. • EGF receptor transactivation is supposed to mediate GPCR
Role of ErbB receptors in cancer cell migration and invasion
Directory of Open Access Journals (Sweden)
Aline eAppert-Collin
2015-11-01
Full Text Available Growth factors mediate their diverse biologic responses (regulation of cellular proliferation, differentiation, migration and survival by binding to and activating cell-surface receptors with intrinsic protein kinase activity named Receptor Tyrosine Kinases (RTKs. About 60 RTKs have been identified and can be classified into more than 16 different receptor families. Their activity is normally tightly controlled and regulated. Overexpression of RTK proteins or functional alterations caused by mutations in the corresponding genes or abnormal stimulation by autocrine growth factor loops contribute to constitutive RTK signaling, resulting in alterations in the physiological activities of cells. The ErbB receptor family of RTKs comprises four distinct receptors: the EGFR (also known as ErbB1/HER1, ErbB2 (neu, HER2, ErbB3 (HER3 and ErbB4 (HER4. ErbB family members are often overexpressed, amplified, or mutated in many forms of cancer, making them important therapeutic targets. EGFR has been found to be amplified in gliomas and non-small-cell lung carcinoma while ErbB2 amplifications are seen in breast, ovarian, bladder, non-small-cell lung carcinoma, as well as several other tumor types. Several data have shown that ErbB receptor family and its downstream pathway regulate epithelial-mesenchymal transition, migration, and tumor invasion by modulating extracellular matrix components. Recent findings indicate that extracellular matrix components such as matrikines bind specifically to EGF receptor and promote cell invasion. In this review, we will present an in-depth overview of the structure, mechanisms, cell signaling, and functions of ErbB family receptors in cell adhesion and migration. Furthermore, we will describe in a last part the new strategies developed in anti-cancer therapy to inhibit ErbB family receptor activation.
Hydrogels with precisely controlled integrin activation dictate vascular patterning and permeability
Li, Shuoran; Nih, Lina R.; Bachman, Haylee; Fei, Peng; Li, Yilei; Nam, Eunwoo; Dimatteo, Robert; Carmichael, S. Thomas; Barker, Thomas H.; Segura, Tatiana
2017-09-01
Integrin binding to bioengineered hydrogel scaffolds is essential for tissue regrowth and regeneration, yet not all integrin binding can lead to tissue repair. Here, we show that through engineering hydrogel materials to promote α3/α5β1 integrin binding, we can promote the formation of a space-filling and mature vasculature compared with hydrogel materials that promote αvβ3 integrin binding. In vitro, α3/α5β1 scaffolds promoted endothelial cells to sprout and branch, forming organized extensive networks that eventually reached and anastomosed with neighbouring branches. In vivo, α3/α5β1 scaffolds delivering vascular endothelial growth factor (VEGF) promoted non-tortuous blood vessel formation and non-leaky blood vessels by 10 days post-stroke. In contrast, materials that promote αvβ3 integrin binding promoted endothelial sprout clumping in vitro and leaky vessels in vivo. This work shows that precisely controlled integrin activation from a biomaterial can be harnessed to direct therapeutic vessel regeneration and reduce VEGF-induced vascular permeability in vivo.
Directory of Open Access Journals (Sweden)
Kiashanee Moodley
Full Text Available The non-integrin laminin receptor, here designated the 37-kDa/67-kDa laminin receptor (LRP/LR, is involved in many physiologically relevant processes, as well as numerous pathological conditions. The overexpression of LRP/LR on various cancerous cell lines plays critical roles in tumour metastasis and angiogenesis. This study investigated whether LRP/LR is implicated in the maintenance of cellular viability in lung and cervical cancer cell lines. Here we show a significant reduction in cellular viability in the aforementioned cell lines as a result of the siRNA-mediated downregulation of LRP. This reduction in cellular viability is due to increased apoptotic processes, reflected by the loss of nuclear integrity and the significant increase in the activity of caspase-3. These results indicate that LRP/LR is involved in the maintenance of cellular viability in tumorigenic lung and cervix uteri cells through the blockage of apoptosis. Knockdown of LRP/LR by siRNA might represent an alternative therapeutic strategy for the treatment of lung and cervical cancer.
International Nuclear Information System (INIS)
Lee, Minyoung; Park, Jung-Jin; Ko, Young-Gyu; Lee, Yun-Sil
2012-01-01
Previously, we found that β-galactoside α2,6-sialyltransferase (ST6Gal I), an enzyme that adds sialic acids to N-linked oligosaccharides of glycoproteins and is frequently overexpressed in cancer cells, is up-regulated by ionizing radiation (IR) and cleaved to a form possessing catalytic activity comparable to that of the Golgi-localized enzyme. Moreover, this soluble form is secreted into the culture media. Induction of ST6Gal I significantly increased the migration of colon cancer cells via sialylation of integrin β1. Here, we further investigated the mechanisms underlying ST6Gal I cleavage, solubilization and release from cells, and addressed its functions, focusing primarily on cancer cell migration. We performed immunoblotting and lectin affinity assay to analyze the expression of ST6 Gal I and level of sialylated integrin β1. After ionizing radiation, migration of cells was measured by in vitro migration assay. α2, 6 sialylation level of cell surface was analyzed by flow cytometry. Cell culture media were concentrated and then analyzed for soluble ST6Gal I levels using an α2, 6 sialyltransferase sandwich ELISA. We found that ST6Gal I was cleaved by BACE1 (β-site amyloid precursor protein-cleaving enzyme), which was specifically overexpressed in response to IR. The soluble form of ST6Gal I, which also has sialyltransferase enzymatic activity, was cleaved from the Golgi membrane and then released into the culture media. Both non-cleaved and cleaved forms of ST6Gal I significantly increased colon cancer cell migration in a sialylation-dependent manner. The pro-migratory effect of the non-cleaved form of ST6Gal I was dependent on integrin β1 sialylation, whereas that of the cleaved form of ST6Gal I was not, suggesting that other intracellular sialylated molecules apart from cell surface molecules such as integrin β1 might be involved in mediating the pro-migratory effects of the soluble form of ST6Gal I. Moreover, production of soluble form ST6Gal I by
International Nuclear Information System (INIS)
Ahmad, Aftab; Schaack, Jerome B.; White, Carl W.; Ahmad, Shama
2013-01-01
Highlights: •A 2A receptor-induced pulmonary endothelial growth is mediated by PI3K and ERK1/2. •Cytosolic calcium mobilization is also critical for pulmonary endothelial growth. •Effectors of A 2A receptor, like tyrosine kinases and cAMP increase PI3K/Akt signaling. •Activation of A 2A receptor can contribute to vascular remodeling. -- Abstract: Hypoxia and HIF-2α-dependent A 2A receptor expression and activation increase proliferation of human lung microvascular endothelial cells (HLMVECs). This study was undertaken to investigate the signaling mechanisms that mediate the proliferative effects of A 2A receptor. A 2A receptor-mediated proliferation of HLMVECs was inhibited by intracellular calcium chelation, and by specific inhibitors of ERK1/2 and PI3-kinase (PI3K). The adenosine A 2A receptor agonist CGS21680 caused intracellular calcium mobilization in controls and, to a greater extent, in A 2A receptor-overexpressing HLMVECs. Adenoviral-mediated A 2A receptor overexpression as well as receptor activation by CGS21680 caused increased PI3K activity and Akt phosphorylation. Cells overexpressing A 2A receptor also manifested enhanced ERK1/2 phosphorylation upon CGS21680 treatment. A 2A receptor activation also caused enhanced cAMP production. Likewise, treatment with 8Br-cAMP increased PI3K activity. Hence A 2A receptor-mediated cAMP production and PI3K and Akt phosphorylation are potential mediators of the A 2A -mediated proliferative response of HLMVECs. Cytosolic calcium mobilization and ERK1/2 phosphorylation are other critical effectors of HLMVEC proliferation and growth. These studies underscore the importance of adenosine A 2A receptor in activation of survival and proliferative pathways in pulmonary endothelial cells that are mediated through PI3K/Akt and ERK1/2 pathways
Chen, S-C; Huang, F-M; Lee, S-S; Li, M-Z; Chang, Y-C
2009-04-01
To investigate the receptor activator of nuclear factor-kappa B (NF-kappaB) ligand (RANKL) in osteoblastic cells stimulated with inflammatory mediators. The expression of RANKL in human osteoblastic cell line U2OS stimulated by pro-inflammatory cytokine interleukin (IL)-1alpha and black-pigmented bacteria Porphyromonas endodontalis was investigated by Western blot and enzyme-linked immunosorbent assay (ELISA). The significance of the results obtained from control and treated groups was statistically analysed by the paired Student's t-test. IL-1alpha was found to upregulate RANKL production in U2OS cells (P endodontalis also increased RANKL expression in U2OS cells after 4-h incubation period demonstrated by Western blot and ELISA (P endodontalis may be involved in developing apical periodontitis through the stimulation of RANKL production.
Kinin B1 receptors contributes to acute pain following minor surgery in humans
Directory of Open Access Journals (Sweden)
Brahim Jaime S
2010-02-01
Full Text Available Abstract Background Kinins play an important role in regulation of pain and hyperalgesia after tissue injury and inflammation by activating two types of G-protein-coupled receptors, the kinin B1 and B2 receptors. It is generally accepted that the B2 receptor is constitutively expressed, whereas the B1 receptor is induced in response to inflammation. However, little is known about the regulatory effects of kinin receptors on the onset of acute inflammation and inflammatory pain in humans. The present study investigated the changes in gene expression of kinin receptors and the levels of their endogenous ligands at an early time point following tissue injury and their relation to clinical pain, as well as the effect of COX-inhibition on their expression levels. Results Tissue injury resulted in a significant up-regulation in the gene expression of B1 and B2 receptors at 3 hours post-surgery, the onset of acute inflammatory pain. Interestingly, the up-regulation in the gene expression of B1 and B2 receptors was positively correlated to pain intensity only after ketorolac treatment, signifying an interaction between prostaglandins and kinins in the inflammatory pain process. Further, the gene expression of both B1 and B2 receptors were correlated. Following tissue injury, B1 ligands des-Arg9-BK and des-Arg10-KD were significantly lower at the third hour compared to the first 2 hours in both the placebo and the ketorolac treatment groups but did not differ significantly between groups. Tissue injury also resulted in the down-regulation of TRPV1 gene expression at 3 hours post-surgery with no significant effect by ketorolac treatment. Interestingly, the change in gene expression of TRPV1 was correlated to the change in gene expression of B1 receptor but not B2 receptor. Conclusions These results provide evidence at the transcriptional level in a clinical model of tissue injury that up-regulation of kinin receptors are involved in the development of the
Host factors that modify Plasmodium falciparum adhesion to endothelial receptors.
Mahamar, Almahamoudou; Attaher, Oumar; Swihart, Bruce; Barry, Amadou; Diarra, Bacary S; Kanoute, Moussa B; Cisse, Kadidia B; Dembele, Adama B; Keita, Sekouba; Gamain, Benoît; Gaoussou, Santara; Issiaka, Djibrilla; Dicko, Alassane; Duffy, Patrick E; Fried, Michal
2017-10-24
P. falciparum virulence is related to adhesion and sequestration of infected erythrocytes (IE) in deep vascular beds, but the endothelial receptors involved in severe malaria remain unclear. In the largest ever study of clinical isolates, we surveyed adhesion of freshly collected IE from children under 5 years of age in Mali to identify novel vascular receptors, and examined the effects of host age, hemoglobin type, blood group and severe malaria on levels of IE adhesion to a panel of endothelial receptors. Several novel molecules, including integrin α3β1, VE-cadherin, ICAM-2, junctional adhesion molecule-B (JAM-B), laminin, and cellular fibronectin, supported binding of IE from children. Severe malaria was not significantly associated with levels of IE adhesion to any of the 19 receptors. Hemoglobin AC, which reduces severe malaria risk, reduced IE binding to the receptors CD36 and integrin α5β1, while hemoglobin AS did not modify IE adhesion to any receptors. Blood groups A, AB and B significantly reduced IE binding to ICAM-1. Severe malaria risk varies with age, but age significantly impacted the level of IE binding to only a few receptors: IE binding to JAM-B decreased with age, while binding to CD36 and integrin α5β1 significantly increased with age.
CCM proteins control endothelial β1 integrin dependent response to shear stress
Directory of Open Access Journals (Sweden)
Zuzana Macek Jilkova
2014-11-01
Full Text Available Hemodynamic shear stress from blood flow on the endothelium critically regulates vascular function in many physiological and pathological situations. Endothelial cells adapt to shear stress by remodeling their cytoskeletal components and subsequently by changing their shape and orientation. We demonstrate that β1 integrin activation is critically controlled during the mechanoresponse of endothelial cells to shear stress. Indeed, we show that overexpression of the CCM complex, an inhibitor of β1 integrin activation, blocks endothelial actin rearrangement and cell reorientation in response to shear stress similarly to β1 integrin silencing. Conversely, depletion of CCM2 protein leads to an elongated “shear-stress-like” phenotype even in the absence of flow. Taken together, our findings reveal the existence of a balance between positive extracellular and negative intracellular signals, i.e. shear stress and CCM complex, for the control of β1 integrin activation and subsequent adaptation of vascular endothelial cells to mechanostimulation by fluid shear stress.
Modulation of β-catenin signaling by glucagon receptor activation.
Directory of Open Access Journals (Sweden)
Jiyuan Ke
Full Text Available The glucagon receptor (GCGR is a member of the class B G protein-coupled receptor family. Activation of GCGR by glucagon leads to increased glucose production by the liver. Thus, glucagon is a key component of glucose homeostasis by counteracting the effect of insulin. In this report, we found that in addition to activation of the classic cAMP/protein kinase A (PKA pathway, activation of GCGR also induced β-catenin stabilization and activated β-catenin-mediated transcription. Activation of β-catenin signaling was PKA-dependent, consistent with previous reports on the parathyroid hormone receptor type 1 (PTH1R and glucagon-like peptide 1 (GLP-1R receptors. Since low-density-lipoprotein receptor-related protein 5 (Lrp5 is an essential co-receptor required for Wnt protein mediated β-catenin signaling, we examined the role of Lrp5 in glucagon-induced β-catenin signaling. Cotransfection with Lrp5 enhanced the glucagon-induced β-catenin stabilization and TCF promoter-mediated transcription. Inhibiting Lrp5/6 function using Dickkopf-1(DKK1 or by expression of the Lrp5 extracellular domain blocked glucagon-induced β-catenin signaling. Furthermore, we showed that Lrp5 physically interacted with GCGR by immunoprecipitation and bioluminescence resonance energy transfer assays. Together, these results reveal an unexpected crosstalk between glucagon and β-catenin signaling, and may help to explain the metabolic phenotypes of Lrp5/6 mutations.
Function of Etk in Growth Factor Receptor Signaling to Integrins in Breast Cancer
National Research Council Canada - National Science Library
Shimizu, Yoji
2001-01-01
The central hypothesis in this IDEA Award is that increased integrin-mediated adhesiveness and migration of breast cancer cells in response to stimulation by the growth factors heregulin beta (HRG beta...
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Patrizia Puddu
Full Text Available Lactoferrin (LF, a key element in mammalian immune system, plays pivotal roles in host defence against infection and excessive inflammation. Its protective effects range from direct antimicrobial activities against a large panel of microbes, including bacteria, viruses, fungi and parasites, to antinflammatory and anticancer activities. In this study, we show that monocyte-derived dendritic cells (MD-DCs generated in the presence of bovine LF (bLF fail to undergo activation by up-modulating CD83, co-stimulatory and major histocompatibility complex molecules, and cytokine/chemokine secretion. Moreover, these cells are weak activators of T cell proliferation and retain antigen uptake activity. Consistent with an impaired maturation, bLF-MD-DC primed T lymphocytes exhibit a functional unresponsiveness characterized by reduced expression of CD154 and impaired expression of IFN-γ and IL-2. The observed imunosuppressive effects correlate with an increased expression of molecules with negative regulatory functions (i.e. immunoglobulin-like transcript 3 and programmed death ligand 1, indoleamine 2,3-dioxygenase, and suppressor of cytokine signaling-3. Interestingly, bLF-MD-DCs produce IL-6 and exhibit constitutive signal transducer and activator of transcription 3 activation. Conversely, bLF exposure of already differentiated MD-DCs completely fails to induce IL-6, and partially inhibits Toll-like receptor (TLR agonist-induced activation. Cell-specific differences in bLF internalization likely account for the distinct response elicited by bLF in monocytes versus immature DCs, providing a mechanistic base for its multiple effects. These results indicate that bLF exerts a potent anti-inflammatory activity by skewing monocyte differentiation into DCs with impaired capacity to undergo activation and to promote Th1 responses. Overall, these bLF-mediated effects may represent a strategy to block excessive DC activation upon TLR-induced inflammation, adding
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Nam, Jin-Min; Onodera, Yasuhito; Bissell, Mina J; Park, Catherine C
2010-04-07
Tactics to selectively enhance cancer radioresponse are of great interest. Cancer cells actively elaborate and remodel their extracellular matrix (ECM) to aid in survival and progression. Previous work has shown that {beta}1-integrin inhibitory antibodies can enhance the growth-inhibitory and apoptotic responses of human breast cancer cell lines to ionizing radiation, either when cells are cultured in three-dimensional laminin-rich ECM (3D lrECM) or grown as xenografts in mice. Here, we show that a specific {alpha} heterodimer of {beta}1-integrin preferentially mediates a prosurvival signal in human breast cancer cells that can be specifically targeted for therapy. 3D lrECM culture conditions were used to compare {alpha}-integrin heterodimer expression in malignant and nonmalignant cell lines. Under these conditions, we found that expression of {alpha}5{beta}1-integrin was upregulated in malignant cells compared with nonmalignant breast cells. Similarly, we found that normal and oncofetal splice variants of fibronectin, the primary ECM ligand of {alpha}5{beta}1-integrin, were also strikingly upregulated in malignant cell lines compared with nonmalignant acini. Cell treatment with a peptide that disrupts the interactions of {alpha}5{beta}1-integrin with fibronectin promoted apoptosis in malignant cells and further heightened the apoptotic effects of radiation. In support of these results, an analysis of gene expression array data from breast cancer patients revealed an association of high levels of {alpha}5-integrin expression with decreased survival. Our findings offer preclinical validation of fibronectin and {alpha}5{beta}1-integrin as targets for breast cancer therapy.
Pistis, M; Muntoni, A L; Pillolla, G; Perra, S; Cignarella, G; Melis, M; Gessa, G L
2005-01-01
Gamma-hydroxybutyric acid (GHB) is a short-chain fatty acid naturally occurring in the mammalian brain, which recently emerged as a major recreational drug of abuse. GHB has multiple neuronal mechanisms including activation of both the GABA(B) receptor, and a distinct GHB-specific receptor. This complex GHB-GABA(B) receptor interaction is probably responsible for the multifaceted pharmacological, behavioral and toxicological profile of GHB. Drugs of abuse exert remarkably similar effects upon reward-related circuits, in particular the mesolimbic dopaminergic system and the nucleus accumbens (NAc). We used single unit recordings in vivo from urethane-anesthetized rats to characterize the effects of GHB on evoked firing in NAc "shell" neurons and on spontaneous activity of antidromically identified dopamine (DA) cells located in the ventral tegmental area. GHB was studied in comparison with the GABA(B) receptor agonist baclofen and antagonist (2S)(+)-5,5-dimethyl-2-morpholineacetic acid (SCH50911). Additionally, we utilized a GHB analog, gamma-(p-methoxybenzil)-gamma-hydroxybutyric acid (NCS-435), devoid of GABA(B) binding properties, but with high affinity for specific GHB binding sites. In common with other drugs of abuse, GHB depressed firing in NAc neurons evoked by the stimulation of the basolateral amygdala. On DA neurons, GHB exerted heterogeneous effects, which were correlated to the baseline firing rate of the cells but led to a moderate stimulation of the DA system. All GHB actions were mediated by GABA(B) receptors, since they were blocked by SCH50911 and were not mimicked by NCS-435. Our study indicates that the electrophysiological profile of GHB is close to typical drugs of abuse: both inhibition of NAc neurons and moderate to strong stimulation of DA transmission are distinctive features of diverse classes of abused drugs. Moreover, it is concluded that addictive and rewarding properties of GHB do not necessarily involve a putative high affinity GHB
Peters, M.E.W.J.; Wendholt, D.; Strietholt, S.; Frank, S.; Pundt, N.; Korb-Pap, A.; Joosten, L.A.B.; Berg, W.B. van den; Kollias, G.; Eckes, B.; Pap, T.
2012-01-01
OBJECTIVE: Integrin alpha2beta1 functions as a major receptor for type I collagen on different cell types, including fibroblasts and inflammatory cells. Although in vitro data suggest a role for alpha2beta1 integrin in regulating both cell attachment and expression of matrix-degrading enzymes such
Vanhaelewyn, Lucas; Schumacher, Paolo; Poelman, Dirk; Fankhauser, Christian; Van Der Straeten, Dominique; Vandenbussche, Filip
2016-11-01
Ultraviolet B (UV-B) light is a part of the solar radiation which has significant effects on plant morphology, even at low doses. In Arabidopsis, many of these morphological changes have been attributed to a specific UV-B receptor, UV resistance locus 8 (UVR8). Recent findings showed that next to phototropin regulated phototropism, UVR8 mediated signaling is able of inducing directional bending towards UV-B light in etiolated seedlings of Arabidopsis, in a phototropin independent manner. In this study, kinetic analysis of phototropic bending was used to evaluate the relative contribution of each of these pathways in UV-B mediated phototropism. Diminishing UV-B light intensity favors the importance of phototropins. Molecular and genetic analyses suggest that UV-B is capable of inducing phototropin signaling relying on phototropin kinase activity and regulation of NPH3. Moreover, enhanced UVR8 responses in the UV-B hypersensitive rup1rup2 mutants interferes with the fast phototropin mediated phototropism. Together the data suggest that phototropins are the most important receptors for UV-B induced phototropism in etiolated seedlings, and a RUP mediated negative feedback pathway prevents UVR8 signaling to interfere with the phototropin dependent response. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.
Directory of Open Access Journals (Sweden)
Harri Lempiäinen
2011-03-01
Full Text Available Evidence suggests that epigenetic perturbations are involved in the adverse effects associated with some drugs and toxicants, including certain classes of non-genotoxic carcinogens. Such epigenetic changes (altered DNA methylation and covalent histone modifications may take place at the earliest stages of carcinogenesis and their identification holds great promise for biomedical research. Here, we evaluate the sensitivity and specificity of genome-wide epigenomic and transcriptomic profiling in phenobarbital (PB-treated B6C3F1 mice, a well-characterized rodent model of non-genotoxic liver carcinogenesis. Methylated DNA Immunoprecipitation (MeDIP-coupled microarray profiling of 17,967 promoter regions and 4,566 intergenic CpG islands was combined with genome-wide mRNA expression profiling to identify liver tissue-specific PB-mediated DNA methylation and transcriptional alterations. Only a limited number of significant anti-correlations were observed between PB-induced transcriptional and promoter-based DNA methylation perturbations. However, the constitutive androstane receptor (CAR target gene Cyp2b10 was found to be concomitantly hypomethylated and transcriptionally activated in a liver tissue-specific manner following PB treatment. Furthermore, analysis of active and repressive histone modifications using chromatin immunoprecipitation revealed a strong PB-mediated epigenetic switch at the Cyp2b10 promoter. Our data reveal that PB-induced transcriptional perturbations are not generally associated with broad changes in the DNA methylation status at proximal promoters and suggest that the drug-inducible CAR pathway regulates an epigenetic switch from repressive to active chromatin at the target gene Cyp2b10. This study demonstrates the utility of integrated epigenomic and transcriptomic profiling for elucidating early mechanisms and biomarkers of non-genotoxic carcinogenesis.
Lempiäinen, Harri; Müller, Arne; Brasa, Sarah; Teo, Soon-Siong; Roloff, Tim-Christoph; Morawiec, Laurent; Zamurovic, Natasa; Vicart, Axel; Funhoff, Enrico; Couttet, Philippe; Schübeler, Dirk; Grenet, Olivier; Marlowe, Jennifer; Moggs, Jonathan; Terranova, Rémi
2011-03-24
Evidence suggests that epigenetic perturbations are involved in the adverse effects associated with some drugs and toxicants, including certain classes of non-genotoxic carcinogens. Such epigenetic changes (altered DNA methylation and covalent histone modifications) may take place at the earliest stages of carcinogenesis and their identification holds great promise for biomedical research. Here, we evaluate the sensitivity and specificity of genome-wide epigenomic and transcriptomic profiling in phenobarbital (PB)-treated B6C3F1 mice, a well-characterized rodent model of non-genotoxic liver carcinogenesis. Methylated DNA Immunoprecipitation (MeDIP)-coupled microarray profiling of 17,967 promoter regions and 4,566 intergenic CpG islands was combined with genome-wide mRNA expression profiling to identify liver tissue-specific PB-mediated DNA methylation and transcriptional alterations. Only a limited number of significant anti-correlations were observed between PB-induced transcriptional and promoter-based DNA methylation perturbations. However, the constitutive androstane receptor (CAR) target gene Cyp2b10 was found to be concomitantly hypomethylated and transcriptionally activated in a liver tissue-specific manner following PB treatment. Furthermore, analysis of active and repressive histone modifications using chromatin immunoprecipitation revealed a strong PB-mediated epigenetic switch at the Cyp2b10 promoter. Our data reveal that PB-induced transcriptional perturbations are not generally associated with broad changes in the DNA methylation status at proximal promoters and suggest that the drug-inducible CAR pathway regulates an epigenetic switch from repressive to active chromatin at the target gene Cyp2b10. This study demonstrates the utility of integrated epigenomic and transcriptomic profiling for elucidating early mechanisms and biomarkers of non-genotoxic carcinogenesis.
Brasa, Sarah; Teo, Soon-Siong; Roloff, Tim-Christoph; Morawiec, Laurent; Zamurovic, Natasa; Vicart, Axel; Funhoff, Enrico; Couttet, Philippe; Schübeler, Dirk; Grenet, Olivier; Marlowe, Jennifer; Moggs, Jonathan; Terranova, Rémi
2011-01-01
Evidence suggests that epigenetic perturbations are involved in the adverse effects associated with some drugs and toxicants, including certain classes of non-genotoxic carcinogens. Such epigenetic changes (altered DNA methylation and covalent histone modifications) may take place at the earliest stages of carcinogenesis and their identification holds great promise for biomedical research. Here, we evaluate the sensitivity and specificity of genome-wide epigenomic and transcriptomic profiling in phenobarbital (PB)-treated B6C3F1 mice, a well-characterized rodent model of non-genotoxic liver carcinogenesis. Methylated DNA Immunoprecipitation (MeDIP)-coupled microarray profiling of 17,967 promoter regions and 4,566 intergenic CpG islands was combined with genome-wide mRNA expression profiling to identify liver tissue-specific PB-mediated DNA methylation and transcriptional alterations. Only a limited number of significant anti-correlations were observed between PB-induced transcriptional and promoter-based DNA methylation perturbations. However, the constitutive androstane receptor (CAR) target gene Cyp2b10 was found to be concomitantly hypomethylated and transcriptionally activated in a liver tissue-specific manner following PB treatment. Furthermore, analysis of active and repressive histone modifications using chromatin immunoprecipitation revealed a strong PB-mediated epigenetic switch at the Cyp2b10 promoter. Our data reveal that PB-induced transcriptional perturbations are not generally associated with broad changes in the DNA methylation status at proximal promoters and suggest that the drug-inducible CAR pathway regulates an epigenetic switch from repressive to active chromatin at the target gene Cyp2b10. This study demonstrates the utility of integrated epigenomic and transcriptomic profiling for elucidating early mechanisms and biomarkers of non-genotoxic carcinogenesis. PMID:21455306
Integrins and small GTPases as modulators of phagocytosis.
Sayedyahossein, Samar; Dagnino, Lina
2013-01-01
Phagocytosis is the mechanism whereby cells engulf large particles. This process has long been recognized as a critical component of the innate immune response, which constitutes the organism's defense against microorganisms. In addition, phagocytic internalization of apoptotic cells or cell fragments plays important roles in tissue homeostasis and remodeling. Phagocytosis requires target interactions with receptors on the plasma membrane of the phagocytic cell. Integrins have been identified as important mediators of particle clearance, in addition to their well-established roles in cell adhesion, migration and mechanotransduction. Indeed, these ubiquitously expressed proteins impart phagocytic capacity to epithelial, endothelial and mesenchymal cell types. The importance of integrins in particle internalization is emphasized by the ability of microbial and viral pathogens to exploit their signaling pathways to invade host cells, and by the wide variety of disorders that arise from abnormalities in integrin-dependent phagocytic uptake. Copyright © 2013 Elsevier Inc. All rights reserved.
Duque, Hernando; LaRocco, Michael; Golde, William T; Baxt, Barry
2004-09-01
At least four members of the integrin family of receptors, alphaVbeta1, alphaVbeta3, alphaVbeta6, and alphaVbeta8, have been identified as receptors for foot-and-mouth disease virus (FMDV) in vitro. Our investigators have recently shown that the efficiency of receptor usage appears to be related to the viral serotype and may be influenced by structural differences on the viral surface (H. Duque and B. Baxt, J. Virol. 77:2500-2511, 2003). To further examine these differences, we generated soluble alphaVbeta3 and alphaVbeta6 integrins. cDNA plasmids encoding the individual complete integrin alphaV, beta3, and beta6 subunits were used to amplify sequences encoding the subunits' signal peptide and ectodomain, resulting in subunits lacking transmembrane and cytoplasmic domains. COS-1 cells were transfected with plasmids encoding the soluble alphaV subunit and either the soluble beta3 or beta6 subunit and labeled with [35S]methionine-cysteine. Complete subunit heterodimeric integrins were secreted into the medium, as determined by radioimmunoprecipitation with specific monoclonal and polyclonal antibodies. For the examination of the integrins' biological activities, stable cell lines producing the soluble integrins were generated in HEK 293A cells. In the presence of divalent cations, soluble alphaVbeta6 bound to representatives of type A or O viruses, immobilized on plastic dishes, and significantly inhibited viral replication, as determined by plaque reduction assays. In contrast, soluble alphaVbeta3 was unable to bind to immobilized virus of either serotype; however, virus bound to the immobilized integrin, suggesting that FMDV binding to alphaVbeta3 is a low-affinity interaction. In addition, soluble alphaVbeta3 did not neutralize virus infectivity. Incubation of soluble alphaVbeta6 with labeled type A12 or O1 resulted in a significant inhibition of virus adsorption to BHK cells, while soluble alphaVbeta3 caused a low (20 to 30%), but consistent, inhibition of virus
Effect of ARA9 on dioxin receptor mediated transcription
International Nuclear Information System (INIS)
Lees, M.J.; Whitelaw, M.L.
2002-01-01
The dioxin (Aryl hydrocarbon) receptor (DR) is a unique bHLH transcription factor which is activated by binding of planar aromatic hydrocarbons typified by dioxin (TCDD). The active receptor is key to metabolism of aryl hydrocarbon xenobiotics by being a potent inducer of CYP1A1 gene activity. Chlorinated dioxins are inert to metabolism and initiate multifarious toxicities, including potent tumour promotion. These ill-effects are mediated by the activated DR and we are studying the mechanisms by which the ligand binding domain of the DR controls activity of the protein. The DR ligand binding domain resides within a PAS (Per/Arnt/Sim homology) region which is contiguous with the bHLH. The latent bHLH/PAS dioxin receptor (DR) is found in the cytoplasm of most mammalian cell types in a complex with heat shock protein 90, a novel immunophilin like protein termed ARA9/XAP2/AIP, and the co-chaperone p23. Here we use antisense ARA9 constructs to reveal that in the absence of ARA9, the DR is unable to form a transcriptionally active complex. Co-expression of antisense ARA9 with a form of the DR which is constitutively targeted to the nucleus leads to dramatically decreased levels of the nuclear DR protein, implying that ARA9 may function beyond its currently proposed role in cytoplasmic retention of the latent DR
Directory of Open Access Journals (Sweden)
Thomas E. Adrian
2008-01-01
Full Text Available Pancreatic cancer is a devastating disease in which current therapies are inadequate. Separate lines of research have identified the 5-lipoxygenase/leukotriene B4 receptor pathway and the PPAR pathway as potential targets for prevention or treatment of this disease. LY293111 was originally designed as a potent leukotriene B4 receptor antagonist for treatment of inflammatory conditions. LY293111 was also known to have inhibitory effects on 5-lipoxygenase, which is upstream of the production of leukotrienes. LY293111 was shown to have potent anticancer effects in pancreatic cancer and several other solid malignancies, where it caused cell cycle arrest and marked apoptosis. Subsequently, it came to light that LY293111 exhibited PPAR agonist activity in addition to its effects on the 5-lipoxygenase pathway. This raises the question of which of the two targets is of greatest importance with regard to the anticancer effects of this agent. The evidence to date is not conclusive, but suggests that the effects of LY293111 may be mediated by both LTB4 receptors and PPAR.
Gaffal, E; Cron, M; Glodde, N; Tüting, T
2013-08-01
∆(9) -Tetrahydrocannabinol (THC), the active constituent of Cannabis sativa, exerts its biological effects in part through the G-protein-coupled CB1 and CB2 receptors, which were initially discovered in brain and spleen tissue, respectively. However, THC also has CB1/2 receptor-independent effects. Because of its immune-inhibitory potential, THC and related cannabinoids are being considered for the treatment of inflammatory skin diseases. Here we investigated the mechanism of the anti-inflammatory activity of THC and the role of CB1 and CB2 receptors. We evaluated the impact of topically applied THC on DNFB-mediated allergic contact dermatitis in wild-type and CB1/2 receptor-deficient mice. We performed immunohistochemical analyses for infiltrating immune cells and studied the influence of THC on the interaction between T cells, keratinocytes and myeloid immune cells in vitro. Topical THC application effectively decreased contact allergic ear swelling and myeloid immune cell infiltration not only in wild-type but also in CB1/2 receptor-deficient mice. We found that THC (1) inhibited the production of IFNγ by T cells, (2) decreased the production of CCL2 and of IFNγ-induced CCL8 and CXL10 by epidermal keratinocytes and (3) thereby limited the recruitment of myeloid immune cells in vitro in a CB1/2 receptor-independent manner. Topically applied THC can effectively attenuate contact allergic inflammation by decreasing keratinocyte-derived pro-inflammatory mediators that orchestrate myeloid immune cell infiltration independent of CB1/2 receptors. This has important implications for the future development of strategies to harness cannabinoids for the treatment of inflammatory skin diseases. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Kappus, Mojdeh S.; Murphy, Andrew J.; Abramowicz, Sandra; Ntonga, Vusisizwe; Welch, Carrie L.; Tall, Alan R.; Westerterp, Marit
2014-01-01
Liver X receptor (LXR) activators decrease atherosclerosis in mice. LXR activators (1) directly upregulate genes involved in reverse cholesterol transport and (2) exert anti-inflammatory effects mediated by transrepression of nuclear factor-κB target genes. We investigated whether myeloid cell
Simon, D I; Ezratty, A M; Francis, S A; Rennke, H; Loscalzo, J
1993-10-15
Fibrin(ogen) (FGN) is important for hemostasis and wound healing and is cleared from sites of injury primarily by the plasminogen activator system. However, there is emerging evidence in plasminogen activator-deficient transgenic mice that nonplasmin pathways may be important in fibrin(ogen)olysis, as well. Given the proximity of FGN and monocytes within the occlusive thrombus at sites of vascular injury, we considered the possibility that monocytes may play an ancillary role in the degradation and clearance of fibrin. We found that monocytes possess an alternative fibrinolytic pathway that uses the integrin Mac-1, which directly binds and internalizes FGN, resulting in its lysosomal degradation. At 4 degrees C, FGN binds to U937 monocytoid cells in a specific and saturable manner with a kd of 1.8 mumol/L. Binding requires adenosine diphosphate stimulation and is calcium-dependent. At 37 degrees C, FGN and fibrin monomer (FM) are internalized and degraded at rates of 0.37 +/- 0.13 and 0.55 +/- 0.03 microgram/10(6) cells/h by U937 cells, 1.38 +/- 0.02 and 1.20 +/- 0.30 microgram/10(6) cells/h by THP-1 cells, and 2.10 +/- 0.20 and 2.52 +/- 0.18 micrograms/10(6) cells/h by human peripheral blood mononuclear cells, respectively. The serine protease inhibitors, PPACK and aprotinin, and the specific elastase inhibitor, AAPVCK, do not significantly inhibit degradation. However, degradation is inhibited by chloroquine, suggesting that a lysosomal pathway is involved. Factor X, a competitive ligand with FGN for the Mac-1 receptor, also blocks degradation, as does a monoclonal antibody to the alpha-subunit of Mac-1. Autoradiography of radioiodinated, internalized FGN shows that FGN proteolysis by the pathway produces a unique degradation pattern distinct from that observed with plasmin. In a fibrin clot lysis assay, Mac-1-mediated fibrinolysis contributed significantly to total fibrinolysis. In summary, FGN is internalized and degraded by activated human monocytoid cells via
Patsoukis, Nikolaos; Bardhan, Kankana; Weaver, Jessica D; Sari, Duygu; Torres-Gomez, Alvaro; Li, Lequn; Strauss, Laura; Lafuente, Esther M; Boussiotis, Vassiliki A
2017-08-22
Lymphocyte activation requires adhesion to antigen-presenting cells. This is a critical event linking innate and adaptive immunity. Lymphocyte adhesion is accomplished through LFA-1, which must be activated by a process referred to as inside-out integrin signaling. Among the few signaling molecules that have been implicated in inside-out integrin activation in hematopoietic cells are the small guanosine triphosphatase (GTPase) Rap1 and its downstream effector Rap1-interacting molecule (RIAM), a multidomain protein that defined the Mig10-RIAM-lamellipodin (MRL) class of adaptor molecules. Through its various domains, RIAM is a critical node of signal integration for activation of T cells, recruits monomeric and polymerized actin to drive actin remodeling and cytoskeletal reorganization, and promotes inside-out integrin signaling in T cells. As a regulator of inside-out integrin activation, RIAM affects multiple functions of innate and adaptive immunity. The effects of RIAM on cytoskeletal reorganization and integrin activation have implications in cell migration and trafficking of cancer cells. We provide an overview of the structure and interactions of RIAM, and we discuss the implications of RIAM functions in innate and adaptive immunity and cancer. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
Platelet-activating factor receptor agonists mediate xeroderma pigmentosum A photosensitivity.
Yao, Yongxue; Harrison, Kathleen A; Al-Hassani, Mohammed; Murphy, Robert C; Rezania, Samin; Konger, Raymond L; Travers, Jeffrey B
2012-03-16
To date, oxidized glycerophosphocholines (Ox-GPCs) with platelet-activating factor (PAF) activity produced non-enzymatically have not been definitively demonstrated to mediate any known disease processes. Here we provide evidence that these Ox-GPCs play a pivotal role in the photosensitivity associated with the deficiency of the DNA repair protein xeroderma pigmentosum type A (XPA). It should be noted that XPA-deficient cells are known to have decreased antioxidant defenses. These studies demonstrate that treatment of human XPA-deficient fibroblasts with the pro-oxidative stressor ultraviolet B (UVB) radiation resulted in increased reactive oxygen species and PAF receptor (PAF-R) agonistic activity in comparison with gene-corrected cells. The UVB irradiation-generated PAF-R agonists were inhibited by antioxidants. UVB irradiation of XPA-deficient (Xpa-/-) mice also resulted in increased PAF-R agonistic activity and skin inflammation in comparison with control mice. The increased UVB irradiation-mediated skin inflammation and TNF-α production in Xpa-/- mice were blocked by systemic antioxidants and by PAF-R antagonists. Structural characterization of PAF-R-stimulating activity in UVB-irradiated XPA-deficient fibroblasts using mass spectrometry revealed increased levels of sn-2 short-chain Ox-GPCs along with native PAF. These studies support a critical role for PAF-R agonistic Ox-GPCs in the pathophysiology of XPA photosensitivity.
Mobasheri, Ali; Buhrmann, Constanze; Aldinger, Constance; Rad, Jafar Soleimani; Shakibaei, Mehdi
2011-01-01
Objective Interleukin-1β (IL-1β) is a pro-inflammatory cytokine that plays a key role in the pathogenesis of osteoarthritis (OA). Growth factors (GFs) capable of antagonizing the catabolic actions of cytokines may have therapeutic potential in the treatment of OA. Herein, we investigated the potential synergistic effects of insulin-like growth factor (IGF-1) and platelet-derived growth factor (PDGF-bb) on different mechanisms participating in IL-1β-induced activation of nuclear transcription factor-κB (NF-κB) and apoptosis in chondrocytes. Methods Primary chondrocytes were treated with IL-1β to induce dedifferentiation and co-treated with either IGF-1 or/and PDGF-bb and evaluated by immunoblotting and electron microscopy. Results Pretreatment of chondrocytes with IGF-1 or/and PDGF-bb suppressed IL-1β-induced NF-κB activation via inhibition of IκB-α kinase. Inhibition of IκB-α kinase by GFs led to the suppression of IκB-α phosphorylation and degradation, p65 nuclear translocation and NF-κB-regulated gene products involved in inflammation and cartilage degradation (COX-2, MMPs) and apoptosis (caspase-3). GFs or BMS-345541 (specific inhibitor of the IKK) reversed the IL-1β-induced down-regulation of collagen type II, cartilage specific proteoglycans, β1-integrin, Shc, activated MAPKinase, Sox-9 and up-regulation of active caspase-3. Furthermore, the inhibitory effects of IGF-1 or/and PDGF-bb on IL-1β-induced NF-κB activation were sensitive to inhibitors of Src (PP1), PI-3K (wortmannin) and Akt (SH-5), suggesting that the pathway consisting of non-receptor tyrosine kinase (Src), phosphatidylinositol 3-kinase and protein kinase B must be involved in IL-1β signaling. Conclusion The results presented suggest that IGF-1 and PDGF-bb are potent inhibitors of IL-1β-mediated activation of NF-κB and apoptosis in chondrocytes, may be mediated in part through suppression of Src/PI-3K/AKT pathway, which may contribute to their anti-inflammatory effects. PMID
Targeting protease activated receptor-1 with P1pal-12 limits bleomycin-induced pulmonary fibrosis
Lin, Cong; Duitman, Janwillem; Daalhuisen, Joost; ten Brink, Marieke; von der Thüsen, Jan; van der Poll, Tom; Borensztajn, Keren; Spek, C. Arnold
2014-01-01
Idiopathic pulmonary fibrosis is the most devastating fibrotic diffuse parenchymal lung disease which remains refractory to pharmacological therapies. Therefore, novel treatments are urgently required. Protease-activated receptor (PAR)-1 is a G-protein-coupled receptor that mediates critical
Directory of Open Access Journals (Sweden)
Elizabeth Dumas
Full Text Available We reported the expression of angiopoietin Tie2 receptor on human neutrophils and the capacity of angiopoietins (Ang1 and Ang2 to induce pro-inflammatory activities, such as platelet-activating factor synthesis, β2-integrin activation and neutrophil migration. Recently, we observed differential effects between both angiopoietins, namely, the capacity of Ang1, but not Ang2, to promote rapid interleukin-8 synthesis and release, as well as neutrophil viability. Herein, we addressed whether Ang1 and/or Ang2 could modulate the synthesis and release of macrophage inflammatory protein-1β (MIP-1β by neutrophils. Neutrophils were isolated from blood of healthy volunteers; intracellular and extracellular MIP-1β protein concentrations were assessed by ELISA. After 24 hours, the basal intracellular and extracellular MIP-1β protein concentrations were ≈500 and 100 pg/106 neutrophils, respectively. Treatment with Ang1 (10 nM increased neutrophil intracellular and extracellular MIP-1β concentrations by 310 and 388% respectively. Pretreatment with PI3K (LY294002, p38 MAPK (SB203580 and MEK (U0126 inhibitors completely inhibited Ang1-mediated increase of MIP-1β intracellular and extracellular protein levels. Pretreatment with NF-κB complex inhibitors, namely Bay11-7085 and IKK inhibitor VII or with a transcription inhibitor (actinomycin D and protein synthesis inhibitor (cycloheximide, did also abrogate Ang1-mediated increase of MIP-1β intracellular and extracellular protein levels. We validated by RT-qPCR analyses the effect of Ang1 on the induction of MIP-1β mRNA levels. Our study is the first one to report Ang1 capacity to induce MIP-1β gene expression, protein synthesis and release from neutrophils, and that these effects are mediated by PI3K, p38 MAPK and MEK activation and downstream NF-κB activation.
International Nuclear Information System (INIS)
Kreis, Stephanie; Schoenfeld, Hans-Joachim; Melchior, Chantal; Steiner, Beat; Kieffer, Nelly
2005-01-01
Integrin receptors are crucial players in cell adhesion and migration. Identification and characterization of cellular proteins that interact with their short α and β cytoplasmic tails will help to elucidate the molecular mechanisms by which integrins mediate bi-directional signaling across the plasma membrane. Integrin α2β1 is a major collagen receptor but to date, only few proteins have been shown to interact with the α2 cytoplasmic tail or with the α2β1 complex. In order to identify novel binding partners of a α2β1cytoplasmic domain complex, we have generated recombinant GST-fusion proteins, incorporating the leucine zipper heterodimerization cassettes of Jun and Fos. To ascertain proper functionality of the recombinant proteins, interaction with natural binding partners was tested. GST-α2 and GST-Jun α2 bound His-tagged calreticulin while GST-β1 and GST-Fos β1 proteins bound talin. In screening assays for novel binding partners, the immobilized GST-Jun α2/GST-Fos β1 heterodimeric complex, but not the single subunits, interacted specifically with endothelial cell-derived vimentin. Vimentin, an abundant intermediate filament protein, has previously been shown to co-localize with αvβ3-positive focal contacts. Here, we provide evidence that this interaction also occurs with α2β1-enriched focal adhesions and we further show that this association is lost after prolonged adhesion of endothelial cells to collagen
The CB1 Receptor as an Important Mediator of Hedonic Reward Processing
Friemel, Chris M; Zimmer, Andreas; Schneider, Miriam
2014-01-01
The endocannabinoid (ECB) system has emerged recently as a key mediator for reward processing. It is well known that cannabinoids affect appetitive learning processes and can induce reinforcing and rewarding effects. However, the involvement of the ECB system in hedonic aspects of reward-related behavior is not completely understood. With the present study, we investigated the modulatory role of the ECB system on hedonic perception, measured by the pleasure attenuated startle (PAS) paradigm for a palatable food reward. Here, a conditioned odor is thought to induce a pleasant affective state that attenuates an aversive reflex—the acoustic startle response. Modulatory effects of the CB1 receptor antagonist/inverse agonist SR1411716 and the cannabinoid agonist WIN 55 212-2 on PAS were examined in rats. PAS was also measured in CB1 receptor knockout (KO) and wild-type (WT) mice. Pharmacological inhibition as well as the absence of CB1 receptors was found to reduce PAS, whereas WIN 55 212-2 administration increased PAS. Finally, presentation of a conditioned reward cue was found to induce striatal FosB/ΔFosB expression in WT mice, but not in KO mice, indicating a reduced stimulation of reward-related brain regions in conditioned KO mice by odor presentation. We here show that in addition to our previous studies in rats, PAS may also serve as a valuable and suitable measure to assess hedonic processing in mice. Our data further indicate that the ECB system, and in particular CB1 receptor signaling, appears to be highly important for the mediation of hedonic aspects of reward processing. PMID:24718372
Neurokinin-1 receptor activation in globus pallidus
Directory of Open Access Journals (Sweden)
Lei Chen
2009-10-01
Full Text Available The undecapeptide substance P has been demonstrated to modulate neuronal activity in a number of brain regions by acting on neurokinin-1 receptors. Anatomical studies revealed a moderate level of neurokinin-1 receptor in rat globus pallidus. To determine the electrophysiological effects of neurokinin-1 receptor activation in globus pallidus, whole-cell patch-clamp recordings were performed in the present study. Under current-clamp recordings, neurokinin-1 receptor agonist, [Sar9, Met(O211] substance P (SM-SP at 1 μM, depolarized globus pallidus neurons and increased their firing rate. Consistently, SM-SP induced an inward current under voltage-clamp recording. The depolarization evoked by SM-SP persisted in the presence of tetrodotoxin, glutamate and GABA receptor antagonists, indicating its direct postsynaptic effects. The neurokinin-1 receptor antagonist, SR140333B, could block SM-SP-induced depolarization. Further experiments showed that suppression of potassium conductance was the predominant ionic mechanism of SM-SP-induced depolarization. To determine if neurokinin-1 receptor activation exerts any effects on GABAergic and glutamatergic neurotransmission, the action of SM-SP on synaptic currents was studied. SM-SP significantly increased the frequency of spontaneous inhibitory postsynaptic currents, but only induced a transient increase in the frequency of miniature inhibitory postsynaptic currents. No change was observed in both spontaneous and miniature excitatory postsynaptic currents. Based on the direct excitatory effects of SM-SP on pallidal neurons, we hypothesize that neurokinin-1 receptor activation in globus pallidus may be involved in the beneficial effect of substance P in Parkinson’s disease.
Zhang, Y; Chen, M; Venugopal, S; Zhou, Y; Xiang, W; Li, Y-H; Lin, Q; Kini, R M; Chong, Y-S; Ge, R
2011-05-05
Isthmin (ISM) is a 60 kDa secreted-angiogenesis inhibitor that suppresses tumor growth in mouse and disrupts vessel patterning in zebrafish embryos. It selectively binds to alphavbeta5 (αvβ5) integrin on the surface of endothelial cells (ECs), but the mechanism of its antiangiogenic action remains unknown. In this work, we establish that soluble ISM suppresses in vitro angiogenesis and induces EC apoptosis by interacting with its cell surface receptor αvβ5 integrin through a novel 'RKD' motif localized within its adhesion-associated domain in MUC4 and other proteins domain. ISM induces EC apoptosis through integrin-mediated death (IMD) by direct recruitment and activation of caspase-8 without causing anoikis. On the other hand, immobilized ISM loses its antiangiogenic function and instead promotes EC adhesion, survival and migration through αvβ5 integrin by activating focal adhesion kinase (FAK). ISM unexpectedly has both a pro-survival and death-promoting effect on ECs depending on its physical state. This dual function of a single antiangiogenic protein may impact its antiangiogenic efficacy in vivo.
Hierarchy of ADAM12 binding to integrins in tumor cells
DEFF Research Database (Denmark)
Thodeti, Charles Kumar; Fröhlich, Camilla; Nielsen, Christian Kamp
2005-01-01
ADAMs (a disintegrin and metalloprotease) comprise a family of cell surface proteins with protease and cell-binding activities. Using different forms and fragments of ADAM12 as substrates in cell adhesion and spreading assays, we demonstrated that alpha9beta1 integrin is the main receptor for ADA...
Directory of Open Access Journals (Sweden)
Brooke M Steenhard
Full Text Available Alport disease in humans, which usually results in proteinuria and kidney failure, is caused by mutations to the COL4A3, COL4A4, or COL4A5 genes, and absence of collagen α3α4α5(IV networks found in mature kidney glomerular basement membrane (GBM. The Alport mouse harbors a deletion of the Col4a3 gene, which also results in the lack of GBM collagen α3α4α5(IV. This animal model shares many features with human Alport patients, including the retention of collagen α1α2α1(IV in GBMs, effacement of podocyte foot processes, gradual loss of glomerular barrier properties, and progression to renal failure. To learn more about the pathogenesis of Alport disease, we undertook a discovery proteomics approach to identify proteins that were differentially expressed in glomeruli purified from Alport and wild-type mouse kidneys. Pairs of cy3- and cy5-labeled extracts from 5-week old Alport and wild-type glomeruli, respectively, underwent 2-dimensional difference gel electrophoresis. Differentially expressed proteins were digested with trypsin and prepared for mass spectrometry, peptide ion mapping/fingerprinting, and protein identification through database searching. The intermediate filament protein, vimentin, was upregulated ∼2.5 fold in Alport glomeruli compared to wild-type. Upregulation was confirmed by quantitative real time RT-PCR of isolated Alport glomeruli (5.4 fold over wild-type, and quantitative confocal immunofluorescence microscopy localized over-expressed vimentin specifically to Alport podocytes. We next hypothesized that increases in vimentin abundance might affect the basement membrane protein receptors, integrins, and screened Alport and wild-type glomeruli for expression of integrins likely to be the main receptors for GBM type IV collagen and laminin. Quantitative immunofluorescence showed an increase in integrin α1 expression in Alport mesangial cells and an increase in integrin α3 in Alport podocytes. We conclude that
Directory of Open Access Journals (Sweden)
Yingying Cai
Full Text Available Family B G protein-coupled receptors (GPCRs play vital roles in hormone-regulated homeostasis. They are drug targets for metabolic diseases, including type 2 diabetes and osteoporosis. Despite their importance, the signaling mechanisms for family B GPCRs at the molecular level remain largely unexplored due to the challenges in purification of functional receptors in sufficient amount for biophysical characterization. Here, we purified the family B GPCR human glucagon-like peptide-1 (GLP-1 receptor (GLP1R, whose agonists, e.g. exendin-4, are used for the treatment of type 2 diabetes mellitus. The receptor was expressed in HEK293S GnTl- cells using our recently developed protocol. The protocol incorporates the receptor into the native-like lipid environment of reconstituted high density lipoprotein (rHDL particles, also known as nanodiscs, immediately after the membrane solubilization step followed by chromatographic purification, minimizing detergent contact with the target receptor to reduce denaturation and prolonging stabilization of receptor in lipid bilayers without extra steps of reconstitution. This method yielded purified GLP1R in nanodiscs that could bind to GLP-1 and exendin-4 and activate Gs protein. This nanodisc purification method can potentially be a general strategy to routinely obtain purified family B GPCRs in the 10s of microgram amounts useful for spectroscopic analysis of receptor functions and activation mechanisms.
Cai, Yingying; Liu, Yuting; Culhane, Kelly J; DeVree, Brian T; Yang, Yang; Sunahara, Roger K; Yan, Elsa C Y
2017-01-01
Family B G protein-coupled receptors (GPCRs) play vital roles in hormone-regulated homeostasis. They are drug targets for metabolic diseases, including type 2 diabetes and osteoporosis. Despite their importance, the signaling mechanisms for family B GPCRs at the molecular level remain largely unexplored due to the challenges in purification of functional receptors in sufficient amount for biophysical characterization. Here, we purified the family B GPCR human glucagon-like peptide-1 (GLP-1) receptor (GLP1R), whose agonists, e.g. exendin-4, are used for the treatment of type 2 diabetes mellitus. The receptor was expressed in HEK293S GnTl- cells using our recently developed protocol. The protocol incorporates the receptor into the native-like lipid environment of reconstituted high density lipoprotein (rHDL) particles, also known as nanodiscs, immediately after the membrane solubilization step followed by chromatographic purification, minimizing detergent contact with the target receptor to reduce denaturation and prolonging stabilization of receptor in lipid bilayers without extra steps of reconstitution. This method yielded purified GLP1R in nanodiscs that could bind to GLP-1 and exendin-4 and activate Gs protein. This nanodisc purification method can potentially be a general strategy to routinely obtain purified family B GPCRs in the 10s of microgram amounts useful for spectroscopic analysis of receptor functions and activation mechanisms.
Okazaki, Taku; Maeda, Akito; Nishimura, Hiroyuki; Kurosaki, Tomohiro; Honjo, Tasuku
2001-01-01
PD-1 is an immunoreceptor that belongs to the immunoglobulin (Ig) superfamily and contains two tyrosine residues in the cytoplasmic region. Studies on PD-1-deficient mice have shown that PD-1 plays critical roles in establishment and/or maintenance of peripheral tolerance, but the mode of action is totally unknown. To study the molecular mechanism for negative regulation of lymphocytes through the PD-1 receptor, we generated chimeric molecules composed of the IgG Fc receptor type IIB (FcγRIIB) extracellular region and the PD-1 cytoplasmic region and expressed them in a B lymphoma cell line, IIA1.6. Coligation of the cytoplasmic region of PD-1 with the B cell receptor (BCR) in IIA1.6 transformants inhibited BCR-mediated growth retardation, Ca2+ mobilization, and tyrosine phosphorylation of effector molecules, including Igβ, Syk, phospholipase C-γ2 (PLCγ2), and ERK1/2, whereas phosphorylation of Lyn and Dok was not affected. Mutagenesis studies indicated that these inhibitory effects do not require the N-terminal tyrosine in the immunoreceptor tyrosine-based inhibitory motif-like sequence, but do require the other tyrosine residue in the C-terminal tail. This tyrosine was phosphorylated and recruited src homology 2-domain-containing tyrosine phosphatase 2 (SHP-2) on coligation of PD-1 with BCR. These results show that PD-1 can inhibit BCR signaling by recruiting SHP-2 to its phosphotyrosine and dephosphorylating key signal transducers of BCR signaling. PMID:11698646
Environmental phthalate monoesters activate pregnane X receptor-mediated transcription
International Nuclear Information System (INIS)
Hurst, Christopher H.; Waxman, David J.
2004-01-01
Phthalate esters, widely used as plasticizers in the manufacture of products made of polyvinyl chloride, induce reproductive and developmental toxicities in rodents. The mechanism that underlies these effects of phthalate exposure, including the potential role of members of the nuclear receptor superfamily, is not known. The present study investigates the effects of phthalates on the pregnane X receptor (PXR), which mediates the induction of enzymes involved in steroid metabolism and xenobiotic detoxification. The ability of phthalate monoesters to activate PXR-mediated transcription was assayed in a HepG2 cell reporter assay following transfection with mouse PXR (mPXR), human PXR (hPXR), or the hPXR allelic variants V140M, D163G, and A370T. Mono-2-ethylhexyl phthalate (MEHP) increased the transcriptional activity of both mPXR and hPXR (5- and 15-fold, respectively) with EC 50 values of 7-8 μM. mPXR and hPXR were also activated by monobenzyl phthalate (MBzP, up to 5- to 6-fold) but were unresponsive to monomethyl phthalate and mono-n-butyl phthalate (M(n)BP) at the highest concentrations tested (300 μM). hPXR-V140M and hPXR-A370T exhibited patterns of phthalate responses similar to the wild-type receptor. By contrast, hPXR-D163G was unresponsive to all phthalate monoesters tested. Further studies revealed that hPXR-D163G did respond to rifampicin, but required approximately 40-fold higher concentrations than wild-type receptor, suggesting that the ligand-binding domain D163G variant has impaired ligand-binding activity. The responsiveness of PXR to activation by phthalate monoesters demonstrated here suggests that these ubiquitous environmental chemicals may, in part, exhibit their endocrine disruptor activities by altering PXR-regulated steroid hormone metabolism with potential adverse health effects in exposed individuals
The impact of quercetin on wound healing relates to changes in αV and β1 integrin expression.
Doersch, Karen M; Newell-Rogers, M Karen
2017-08-01
Overly fibrotic wound healing can lead to excess scar formation, causing functional impairment and undesirable cosmetic results. However, there are few successful treatments available to prevent or remediate scars. This study sought to explore the molecular mechanisms by which quercetin, a naturally-occurring antifibrotic agent, diminishes scar formation. Using both mice and fibroblast cells, we examined quercetin's impact on fibrosis and the wound healing rate, and potential molecular mechanisms underlying the quercetin-mediated reduction of fibrosis. While cultured fibroblasts demonstrated normal growth in response to quercetin, quercetin increased surface αV integrin and decreased β1 integrin. These changes in surface integrin expression may impact factors that contribute to fibrosis including cell migration, proliferation, and extracellular matrix production. In both quercetin-treated and control mice, wounds healed in about 14 days. Masson's trichrome stain revealed diminished fibrosis at the wound site in quercetin-treated animals despite the normal healing rate, indicating the potential for better cosmetic results without delaying healing. An in vitro scratch wound model using cells plated on an artificial extracellular matrix demonstrated delayed closure following quercetin treatment. The extracellular matrix also ameliorated quercetin's effect on αV integrin. Thus, αV integrin recruitment in response to quercetin treatment may promote the quercetin-mediated decrease extracellular matrix because cells require less extracellular matrix to migrate into a wound. With added extracellular matrix, β1 integrin remained diminished in response to quercetin, indicating that quercetin's effect on β1 integrin expression is independent of extracellular matrix -mediated signaling and is likely driven by inhibition of the intracellular mechanisms driving β1 expression. These findings suggest that quercetin could alter the cells' interactions with the extracellular
Combined sodium ion sensitivity in agonist binding and internalization of vasopressin V1b receptors.
Koshimizu, Taka-Aki; Kashiwazaki, Aki; Taniguchi, Junichi
2016-05-03
Reducing Na(+) in the extracellular environment may lead to two beneficial effects for increasing agonist binding to cell surface G-protein coupled receptors (GPCRs): reduction of Na(+)-mediated binding block and reduce of receptor internalization. However, such combined effects have not been explored. We used Chinese Hamster Ovary cells expressing vasopressin V1b receptors as a model to explore Na(+) sensitivity in agonist binding and receptor internalization. Under basal conditions, a large fraction of V1b receptors is located intracellularly, and a small fraction is in the plasma membrane. Decreases in external Na(+) increased cell surface [(3)H]AVP binding and decreased receptor internalization. Substitution of Na(+) by Cs(+) or NH4(+) inhibited agonist binding. To suppress receptor internalization, the concentration of NaCl, but not of CsCl, had to be less than 50 mM, due to the high sensitivity of the internalization machinery to Na(+) over Cs(+). Iso-osmotic supplementation of glucose or NH4Cl maintained internalization of the V1b receptor, even in a low-NaCl environment. Moreover, iodide ions, which acted as a counter anion, inhibited V1b agonist binding. In summary, we found external ionic conditions that could increase the presence of high-affinity state receptors at the cell surface with minimum internalization during agonist stimulations.
Social Context, Stress, Neuropsychiatric Disorders, and the Vasopressin 1b Receptor
Directory of Open Access Journals (Sweden)
Heather K. Caldwell
2017-10-01
Full Text Available The arginine vasopressin 1b receptor (Avpr1b is involved in the modulation of a variety of behaviors and is an important part of the mammalian hormonal stress axis. The Avpr1b is prominent in hippocampal CA2 pyramidal cells and in the anterior pituitary corticotrophs. Decades of research on this receptor has demonstrated its importance to the modulation of social recognition memory, social forms of aggression, and modulation of the hypothalamic-pituitary-adrenal axis, particularly under conditions of acute stress. Further, work in humans suggests that the Avpr1b may play a role in human neuropsychiatric disorders and its modulation may have therapeutic potential. This paper reviews what is known about the role of the Avpr1b in the context of social behaviors, the stress axis, and human neuropsychiatric disorders. Further, possible mechanisms for how Avpr1b activation within the hippocampus vs. Avpr1b activation within anterior pituitary may interact with one another to affect behavioral output are proposed.
Effects of serotonin (5-HT)1B receptor ligands on amphetamine-seeking behavior in rats.
Miszkiel, Joanna; Przegaliński, Edmund
2013-01-01
Numerous studies have indicated that serotonin (5-HT)1B receptor ligands affect the behavioral effects of psychostimulants (cocaine, amphetamine), including the reinforcing activities of these drugs. To substantiate a role for those receptors in incentive motivation for amphetamine, we used the extinction/reinstatement model to examine the effects of the 5-HT1B receptor ligands on the reinstatement of extinguished amphetamine-seeking behavior. Rats trained to self-administer amphetamine (0.06 mg/kg/infusion) subsequently underwent the extinction procedure. These rats were then tested for the amphetamine-primed or amphetamine-associated cue-induced reinstatement of extinguished amphetamine-seeking behavior. The 5-HT1B receptor antagonist SB 216641 (5-7.5 mg/kg) attenuated the amphetamine (1.5 mg/kg)- and the amphetamine-associated cue combined with the threshold dose of amphetamine (0.5 mg/kg)-induced reinstatement of amphetamine-seeking behavior. The 5-HT1B receptor agonist CP 94253 (1.25-5 mg/kg) also inhibited the amphetamine-seeking behavior induced by amphetamine (1.5 mg/kg) but not by the cue combined with the threshold dose of amphetamine. The inhibitory effect of CP94253 on amphetamine-seeking behavior remained unaffected by the 5-HT1B receptor antagonist. Our results indicate that tonic activation of 5-HT1B receptors is involved in amphetamine- and cue-induced reinstatement of amphetamine-seeking behavior and that the inhibitory effects of 5-HT1B receptor antagonists on these phenomena are directly related to the motivational aspects of amphetamine abuse. The inhibitory effect of CP 94253 on amphetamine-seeking behavior seems to be unrelated to 5-HT1B receptor activation and may result from a general reduction of motivation.
de Graaf, C.; Donnely, D.; Wootten, D.; Lau, J.; Sexton, P.M.; Miller, L.J.; Ahn, J.M.; Liao, J.; Fletcher, M.M.; Yang, D.; Brown, A.J.; Zhou, C.; Deng, J.; Wang, M.W.
2016-01-01
Theglucagon-likepeptide (GLP)-1receptor (GLP-1R) is a class B G protein-coupled receptor (GPCR) that mediates the action of GLP-1, a peptide hormone secretedfromthreemajor tissues inhumans,enteroendocrine L cells in the distal intestine, a cells in the pancreas, and the central nervous system, which
Long-lived, high-strength states of ICAM-1 bonds to beta2 integrin, I
DEFF Research Database (Denmark)
Evans, Evan; Kinoshita, Koji; Simon, Scott
2010-01-01
Using single-molecule force spectroscopy to probe ICAM-1 interactions with recombinant alphaLbeta2 immobilized on microspheres and beta2 integrin on neutrophils, we quantified an impressive hierarchy of long-lived, high-strength states of the integrin bond, which start from basal levels with inte......Using single-molecule force spectroscopy to probe ICAM-1 interactions with recombinant alphaLbeta2 immobilized on microspheres and beta2 integrin on neutrophils, we quantified an impressive hierarchy of long-lived, high-strength states of the integrin bond, which start from basal levels...... with integrin activation in solutions of divalent cations and shift dramatically upward to hyperactivated states with cell signaling in leukocytes. Taking advantage of very rare events, we used repeated measurements of bond lifetimes under steady ramps of force to achieve a direct assay for the off......-based assays of soluble ICAM-1 dissociation from immobilized LFA-1, i.e., approximately 10(-2)/s in Mg2+ or Mn2+ and approximately 1/s in Ca2+. At the same time, as expected for adhesive function, we find that the beta2 integrin bonds activated in Mn2+ or Mg2+ possess significant and persistent mechanical...
Sphingosine-1-phosphate mediates epidermal growth factor-induced muscle satellite cell activation
Energy Technology Data Exchange (ETDEWEB)
Nagata, Yosuke, E-mail: cynagata@mail.ecc.u-tokyo.ac.jp; Ohashi, Kazuya; Wada, Eiji; Yuasa, Yuki; Shiozuka, Masataka; Nonomura, Yoshiaki; Matsuda, Ryoichi
2014-08-01
Skeletal muscle can regenerate repeatedly due to the presence of resident stem cells, called satellite cells. Because satellite cells are usually quiescent, they must be activated before participating in muscle regeneration in response to stimuli such as injury, overloading, and stretch. Although satellite cell activation is a crucial step in muscle regeneration, little is known of the molecular mechanisms controlling this process. Recent work showed that the bioactive lipid sphingosine-1-phosphate (S1P) plays crucial roles in the activation, proliferation, and differentiation of muscle satellite cells. We investigated the role of growth factors in S1P-mediated satellite cell activation. We found that epidermal growth factor (EGF) in combination with insulin induced proliferation of quiescent undifferentiated mouse myoblast C2C12 cells, which are also known as reserve cells, in serum-free conditions. Sphingosine kinase activity increased when reserve cells were stimulated with EGF. Treatment of reserve cells with the D-erythro-N,N-dimethylsphingosine, Sphingosine Kinase Inhibitor, or siRNA duplexes specific for sphingosine kinase 1, suppressed EGF-induced C2C12 activation. We also present the evidence showing the S1P receptor S1P2 is involved in EGF-induced reserve cell activation. Moreover, we demonstrated a combination of insulin and EGF promoted activation of satellite cells on single myofibers in a manner dependent on SPHK and S1P2. Taken together, our observations show that EGF-induced satellite cell activation is mediated by S1P and its receptor. - Highlights: • EGF in combination with insulin induces proliferation of quiescent C2C12 cells. • Sphingosine kinase activity increases when reserve cells are stimulated with EGF. • EGF-induced activation of reserve cells is dependent on sphingosine kinase and ERK. • The S1P receptor S1P2 is involved in EGF-induced reserve cell activation. • EGF-induced reserve cell activation is mediated by S1P and its
Sphingosine-1-phosphate mediates epidermal growth factor-induced muscle satellite cell activation
International Nuclear Information System (INIS)
Nagata, Yosuke; Ohashi, Kazuya; Wada, Eiji; Yuasa, Yuki; Shiozuka, Masataka; Nonomura, Yoshiaki; Matsuda, Ryoichi
2014-01-01
Skeletal muscle can regenerate repeatedly due to the presence of resident stem cells, called satellite cells. Because satellite cells are usually quiescent, they must be activated before participating in muscle regeneration in response to stimuli such as injury, overloading, and stretch. Although satellite cell activation is a crucial step in muscle regeneration, little is known of the molecular mechanisms controlling this process. Recent work showed that the bioactive lipid sphingosine-1-phosphate (S1P) plays crucial roles in the activation, proliferation, and differentiation of muscle satellite cells. We investigated the role of growth factors in S1P-mediated satellite cell activation. We found that epidermal growth factor (EGF) in combination with insulin induced proliferation of quiescent undifferentiated mouse myoblast C2C12 cells, which are also known as reserve cells, in serum-free conditions. Sphingosine kinase activity increased when reserve cells were stimulated with EGF. Treatment of reserve cells with the D-erythro-N,N-dimethylsphingosine, Sphingosine Kinase Inhibitor, or siRNA duplexes specific for sphingosine kinase 1, suppressed EGF-induced C2C12 activation. We also present the evidence showing the S1P receptor S1P2 is involved in EGF-induced reserve cell activation. Moreover, we demonstrated a combination of insulin and EGF promoted activation of satellite cells on single myofibers in a manner dependent on SPHK and S1P2. Taken together, our observations show that EGF-induced satellite cell activation is mediated by S1P and its receptor. - Highlights: • EGF in combination with insulin induces proliferation of quiescent C2C12 cells. • Sphingosine kinase activity increases when reserve cells are stimulated with EGF. • EGF-induced activation of reserve cells is dependent on sphingosine kinase and ERK. • The S1P receptor S1P2 is involved in EGF-induced reserve cell activation. • EGF-induced reserve cell activation is mediated by S1P and its
Targeting ILK and β4 integrin abrogates the invasive potential of ovarian cancer
International Nuclear Information System (INIS)
Choi, Yoon Pyo; Kim, Baek Gil; Gao, Ming-Qing; Kang, Suki; Cho, Nam Hoon
2012-01-01
Highlights: ► The potential of targeting ILK and integrins for highly aggressive ovarian cancer. ► Unanticipated synergistic effect for the combination of ILK/β4 integrin. ► Combination of ILK/β4 integrin effectively inhibited the PI3K/Akt/Rac1 cascade. ► Targeting of β4 integrin/ILK had potent inhibitory effects in ovarian cancer. -- Abstract: Integrins and integrin-linked kinase (ILK) are essential to cancerous invasion because they mediate physical interactions with the extracellular matrix, and regulate oncogenic signaling pathways. The purpose of our study is to determine whether deletion of β1 and β4 integrin and ILK, alone or in combination, has antitumoral effects in ovarian cancer. Expression of β1 and β4 integrin and ILK was analyzed by immunohistochemistry in 196 ovarian cancer tissue samples. We assessed the effects of depleting these molecules with shRNAs in ovarian cancer cells by Western blot, conventional RT-PCR, cell proliferation, migration, invasion, and in vitro Rac1 activity assays, and in vivo xenograft formation assays. Overexpression of β4 integrin and ILK in human ovarian cancer specimens was found to correlate with tumor aggressiveness. Depletion of these targets efficiently suppresses ovarian cancer cell proliferation, migration, and invasion in vitro and xenograft tumor formation in vivo. We also demonstrated that single depletion of ILK or combination depletion of β4 integrin/ILK inhibits phosphorylation of downstream signaling targets, p-Ser 473 Akt and p-Thr202/Tyr204 Erk1/2, and activation of Rac1, as well as reduce expression of MMP-2 and MMP-9 and increase expression of caspase-3 in vitro. In conclusion, targeting β4 integrin combined with ILK can instigate the latent tumorigenic potential and abrogate the invasive potential in ovarian cancer.
Targeting ILK and {beta}4 integrin abrogates the invasive potential of ovarian cancer
Energy Technology Data Exchange (ETDEWEB)
Choi, Yoon Pyo; Kim, Baek Gil [BK21 Project for Medical Science, Yonsei University College of Medicine, Seoul (Korea, Republic of); Department of Pathology, Yonsei University College of Medicine, Seoul (Korea, Republic of); Gao, Ming-Qing; Kang, Suki [Department of Pathology, Yonsei University College of Medicine, Seoul (Korea, Republic of); Cho, Nam Hoon, E-mail: cho1988@yuhs.ac [BK21 Project for Medical Science, Yonsei University College of Medicine, Seoul (Korea, Republic of); Department of Pathology, Yonsei University College of Medicine, Seoul (Korea, Republic of)
2012-10-26
Highlights: Black-Right-Pointing-Pointer The potential of targeting ILK and integrins for highly aggressive ovarian cancer. Black-Right-Pointing-Pointer Unanticipated synergistic effect for the combination of ILK/{beta}4 integrin. Black-Right-Pointing-Pointer Combination of ILK/{beta}4 integrin effectively inhibited the PI3K/Akt/Rac1 cascade. Black-Right-Pointing-Pointer Targeting of {beta}4 integrin/ILK had potent inhibitory effects in ovarian cancer. -- Abstract: Integrins and integrin-linked kinase (ILK) are essential to cancerous invasion because they mediate physical interactions with the extracellular matrix, and regulate oncogenic signaling pathways. The purpose of our study is to determine whether deletion of {beta}1 and {beta}4 integrin and ILK, alone or in combination, has antitumoral effects in ovarian cancer. Expression of {beta}1 and {beta}4 integrin and ILK was analyzed by immunohistochemistry in 196 ovarian cancer tissue samples. We assessed the effects of depleting these molecules with shRNAs in ovarian cancer cells by Western blot, conventional RT-PCR, cell proliferation, migration, invasion, and in vitro Rac1 activity assays, and in vivo xenograft formation assays. Overexpression of {beta}4 integrin and ILK in human ovarian cancer specimens was found to correlate with tumor aggressiveness. Depletion of these targets efficiently suppresses ovarian cancer cell proliferation, migration, and invasion in vitro and xenograft tumor formation in vivo. We also demonstrated that single depletion of ILK or combination depletion of {beta}4 integrin/ILK inhibits phosphorylation of downstream signaling targets, p-Ser 473 Akt and p-Thr202/Tyr204 Erk1/2, and activation of Rac1, as well as reduce expression of MMP-2 and MMP-9 and increase expression of caspase-3 in vitro. In conclusion, targeting {beta}4 integrin combined with ILK can instigate the latent tumorigenic potential and abrogate the invasive potential in ovarian cancer.
The role of receptor-mediated T-cells activation disorders in pulmonary tuberculosis
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Irina E. Esimova
2017-01-01
Full Text Available Aim. To analyze the peculiarities and mechanisms of receptor-mediated T-lymphocytes disorders in different clinical forms of pulmonary tuberculosis.Materials and мethods. The study involved 116 patients with first diagnosed infiltrative and disseminated drug-sensitive and drug-resistant pulmonary tuberculosis. The key stages in receptor-mediated activation of T-lymphocytes, isolated from blood, after their CD3/CD28-induction in vitro with addition of intracellular transport blocker were analyzed. Their immunotyping was carried out with the method of two- and threecolor flow cytofluorometry. The obtained results were statistically analyzed.Results. The breach of extracellular and intracellular stages of T-lymphocytes activation, shown by reduction in total number of CD3- and CD28-positive cells, and CD3+CD28+IL2+, CD3+CD28+IL2–, CD3+NF-kB+, CD3+NFAT2+ lymphocytes, and increase in number of CD3+CTLA4+ cells, was identified with most of their manifestations in disseminated drug-resistant pulmonary tuberculosis. It was shown that the content of CD3+AP-1+ lymphocytes is variable in drug-resistant pulmonary tuberculosis: it increases in the infiltrative form and decreases in the disseminated form.Conclusion. The results showed different mechanisms leading to a deficiency of IL-2-positive lymphocytes and T-lymphocytopenia: from “functional reserve” exhaustion of T-cells in drug-sensitive pulmonary tuberculosis to immunosuppression under the influence of suppressive cytokines (in case of the infiltrative form and inhibitory protein CTLA4 (in case of the disseminated form in drug-resistant pulmonary tuberculosis.
LENUS (Irish Health Repository)
Krause-Gruszczynska, Malgorzata
2011-12-28
Abstract Background Host cell invasion by the foodborne pathogen Campylobacter jejuni is considered as one of the primary reasons of gut tissue damage, however, mechanisms and key factors involved in this process are widely unclear. It was reported that small Rho GTPases, including Cdc42, are activated and play a role during invasion, but the involved signaling cascades remained unknown. Here we utilised knockout cell lines derived from fibronectin-\\/-, integrin-beta1-\\/-, focal adhesion kinase (FAK)-\\/- and Src\\/Yes\\/Fyn-\\/- deficient mice, and wild-type control cells, to investigate C. jejuni-induced mechanisms leading to Cdc42 activation and bacterial uptake. Results Using high-resolution scanning electron microscopy, GTPase pulldowns, G-Lisa and gentamicin protection assays we found that each studied host factor is necessary for induction of Cdc42-GTP and efficient invasion. Interestingly, filopodia formation and associated membrane dynamics linked to invasion were only seen during infection of wild-type but not in knockout cells. Infection of cells stably expressing integrin-beta1 variants with well-known defects in fibronectin fibril formation or FAK signaling also exhibited severe deficiencies in Cdc42 activation and bacterial invasion. We further demonstrated that infection of wild-type cells induces increasing amounts of phosphorylated FAK and growth factor receptors (EGFR and PDGFR) during the course of infection, correlating with accumulating Cdc42-GTP levels and C. jejuni invasion over time. In studies using pharmacological inhibitors, silencing RNA (siRNA) and dominant-negative expression constructs, EGFR, PDGFR and PI3-kinase appeared to represent other crucial components upstream of Cdc42 and invasion. siRNA and the use of Vav1\\/2-\\/- knockout cells further showed that the guanine exchange factor Vav2 is required for Cdc42 activation and maximal bacterial invasion. Overexpression of certain mutant constructs indicated that Vav2 is a linker
Czech Academy of Sciences Publication Activity Database
Vondráček, Jan; Chramostová, Kateřina; Plíšková, M.; Bláha, L.; Brack, W.; Kozubík, Alois; Machala, M.
2004-01-01
Roč. 23, č. 9 (2004), s. 2214-2220 ISSN 0730-7268 R&D Projects: GA ČR GA525/03/1527 Institutional research plan: CEZ:AV0Z5004920 Keywords : aryl hydrocarbon receptor-mediated activity * estrogenicity * intercellular communication inhibition Subject RIV: BO - Biophysics Impact factor: 2.121, year: 2004
Directory of Open Access Journals (Sweden)
Yosuke Masubuchi
Full Text Available We previously reported that 3T3-L1 cells express a functional sweet taste receptor possibly as a T1R3 homomer that is coupled to Gs and negatively regulates adipogenesis by a Gαs-mediated but cAMP-independent mechanism. Here, we show that stimulation of this receptor with sucralose or saccharin induced disassembly of the microtubules in 3T3-L1 preadipocytes, which was attenuated by overexpression of the dominant-negative mutant of Gαs (Gαs-G226A. In contrast, overexpression of the constitutively active mutant of Gαs (Gαs-Q227L as well as treatment with cholera toxin or isoproterenol but not with forskolin caused disassembly of the microtubules. Sweetener-induced microtubule disassembly was accompanied by activation of RhoA and Rho-associated kinase (ROCK. This was attenuated with by knockdown of GEF-H1, a microtubule-localized guanine nucleotide exchange factor for Rho GTPase. Furthermore, overexpression of the dominant-negative mutant of RhoA (RhoA-T19N blocked sweetener-induced dephosphorylation of Akt and repression of PPARγ and C/EBPα in the early phase of adipogenic differentiation. These results suggest that the T1R3 homomeric sweet taste receptor negatively regulates adipogenesis through Gαs-mediated microtubule disassembly and consequent activation of the Rho/ROCK pathway.
Steeghs, Liana; van Vliet, Sandra J; Uronen-Hansson, Heli; van Mourik, Andries; Engering, Anneke; Sanchez-Hernandez, Martha; Klein, Nigel; Callard, Robin; van Putten, Jos P M; van der Ley, Peter; van Kooyk, Yvette; van de Winkel, Jan G J
2006-02-01
Neisseria meningitidis lipopolysaccharide (LPS) has been identified as a major determinant of dendritic cell (DC) function. Here we report that one of a series of meningococcal mutants with defined truncations in the lacto-N-neotetraose outer core of the LPS exhibited unique strong adhesion and internalization properties towards DC. These properties were mediated by interaction of the GlcNAc(beta1-3)-Gal(beta1-4)-Glc-R oligosaccharide outer core of lgtB LPS with the dendritic-cell-specific ICAM-3 grabbing non-integrin (DC-SIGN) lectin receptor. Activation of DC-SIGN with this novel oligosaccharide ligand skewed T-cell responses driven by DC towards T helper type 1 activity. Thus, the use of lgtB LPS may provide a powerful instrument to selectively induce the desired arm of the immune response and potentially increase vaccine efficacy.
Pae, So Hyun; Dokic, Danijela; Dettman, Robert W
2008-04-01
Formation of the epicardium requires interactions between alpha(4)beta(1) integrin, and the extracellular matrix. We investigated the role of other integrins expressed by epicardial cells. We detected transcripts for alpha(5), alpha(8), alpha(v), beta(1), beta(3), and beta(5) integrins in the chick proepicardial organ (PE). We demonstrate that alpha(5)beta(1), alpha(8)beta(1), and alpha(v)beta(3) integrins are expressed by chick epicardial mesothelial cells (EMCs). Migration of EMCs in vitro was reduced by RGD-containing peptides. Using adenoviruses expressing an antisense to chick alpha(4) (AdGFPalpha4AS), full-length (Adhalpha4V5), and C-terminal deleted alpha(4) (Adhalpha4DeltaCV5), we found that EMCs were less able to adhere to vitronectin and fibronectin(120) indicating that alpha(4)beta(1) plays a role in regulating EMC adhesion to ligands of alpha(5)beta(1), alpha(8)beta(1), and alpha(v)beta(3). In Adhalpha4DeltaCV5-infected EMCs, alpha(5)beta(1) was diminished in fibrillar adhesions and new FN matrix assembly was abnormal. We propose that cooperation between alpha(4)beta(1) and RGD integrins is important for EMC adhesion and subepicardial matrix formation. (c) 2008 Wiley-Liss, Inc.
He, Yuan; Kapoor, Ashish; Cook, Sara; Liu, Shubai; Xiang, Yang; Rao, Christopher V.; Kenis, Paul J. A.; Wang, Fei
2011-01-01
Establishing new adhesions at the extended leading edges of motile cells is essential for stable polarity and persistent motility. Despite recent identification of signaling pathways that mediate polarity and chemotaxis in neutrophils, little is known about molecular mechanisms governing cell–extracellular-matrix (ECM) adhesion in these highly polarized and rapidly migrating cells. Here, we describe a signaling pathway in neutrophils that is essential for localized integrin activation, leading edge attachment and persistent migration during chemotaxis. This pathway depends upon Gi-protein-mediated activation and leading edge recruitment of Lyn, a non-receptor tyrosine kinase belonging to the Src kinase family. We identified the small GTPase Rap1 as a major downstream effector of Lyn to regulate neutrophil adhesion during chemotaxis. Depletion of Lyn in neutrophil-like HL-60 cells prevented chemoattractant-induced Rap1 activation at the leading edge of the cell, whereas ectopic expression of Rap1 largely rescued the defects induced by Lyn depletion. Furthermore, Lyn controls spatial activation of Rap1 by recruiting the CrkL–C3G protein complex to the leading edge. Together, these results provide novel mechanistic insights into the poorly understood signaling network that controls leading edge adhesion during chemotaxis of neutrophils, and possibly other amoeboid cells. PMID:21628423
HTLV-1 Tax-mediated TAK1 activation involves TAB2 adapter protein
International Nuclear Information System (INIS)
Yu Qingsheng; Minoda, Yasumasa; Yoshida, Ryoko; Yoshida, Hideyuki; Iha, Hidekatsu; Kobayashi, Takashi; Yoshimura, Akihiko; Takaesu, Giichi
2008-01-01
Human T cell leukemia virus type 1 (HTLV-1) Tax is an oncoprotein that plays a crucial role in the proliferation and transformation of HTLV-1-infected T lymphocytes. It has recently been reported that Tax activates a MAPKKK family, TAK1. However, the molecular mechanism of Tax-mediated TAK1 activation is not well understood. In this report, we investigated the role of TAK1-binding protein 2 (TAB2) in Tax-mediated TAK1 activation. We found that TAB2 physically interacts with Tax and augments Tax-induced NF-κB activity. Tax and TAB2 cooperatively activate TAK1 when they are coexpressed. Furthermore, TAK1 activation by Tax requires TAB2 binding as well as ubiquitination of Tax. We also found that the overexpression of TRAF2, 5, or 6 strongly induces Tax ubiquitination. These results suggest that TAB2 may be critically involved in Tax-mediated activation of TAK1 and that NF-κB-activating TRAF family proteins are potential cellular E3 ubiquitin ligases toward Tax
Yankee, Thomas M; Solow, Sasha A; Draves, Kevin D; Clark, Edward A
2003-01-01
Adapter proteins play a critical role in regulating signals triggered by Ag receptor cross-linking. These small molecules link receptor proximal events with downstream signaling pathways. In this study, we explore the expression and function of the Grb2-related protein of the lymphoid system (GrpL)/Grb2-related adaptor downstream of Shc adapter protein in human B cells. GrpL is expressed in naive B cells and is down-regulated following B cell Ag receptor ligation. By contrast, germinal center and memory B cells express little or no GrpL. Using human B cell lines, we detected constitutive interactions between GrpL and B cell linker protein, Src homology (SH)2 domain-containing leukocyte protein of 76 kDa, hemopoietic progenitor kinase 1, and c-Cbl. The N-terminal SH3 domain of GrpL binds c-Cbl while the C-terminal SH3 domain binds B cell linker protein and SH2 domain-containing leukocyte protein of 76 kDa. Exogenous expression of GrpL in a GrpL-negative B cell line leads to enhanced Ag receptor-induced extracellular signal-related kinase and p38 mitogen-activated protein kinase phosphorylation. Thus, GrpL expression in human B cell subsets appears to regulate Ag receptor-mediated signaling events.
P2X1 receptors and the endothelium
Directory of Open Access Journals (Sweden)
LS Harrington
2005-03-01
Full Text Available Adenosine triphosphate (ATP is now established as a principle vaso-active mediator in the vasculature. Its actions on arteries are complex, and are mediated by the P2X and P2Y receptor families. It is generally accepted that ATP induces a bi-phasic response in arteries, inducing contraction via the P2X and P2Y receptors on the smooth muscle cells, and vasodilation via the actions of P2Y receptors located on the endothelium. However, a number of recent studies have placed P2X1 receptors on the endothelium of some arteries. The use of a specific P2X1 receptor ligand, a, b methylene ATP has demonstrated that P2X1 receptors also have a bi-functional role. The actions of ATP on P2X1 receptors is therefore dependant on its location, inducing contraction when located on the smooth muscle cells, and dilation when expressed on the endothelium, comparable to that of P2Y receptors.
Li, Minghua; Li, Zhiqin; Morris, David L.; Rui, Liangyou
2007-01-01
The SH2B family has three members (SH2B1, SH2B2, and SH2B3) that contain conserved dimerization (DD), pleckstrin homology, and SH2 domains. The DD domain mediates the formation of homo- and heterodimers between members of the SH2B family. The SH2 domain of SH2B1 (previously named SH2-B) or SH2B2 (previously named APS) binds to phosphorylated tyrosines in a variety of tyrosine kinases, including Janus kinase-2 (JAK2) and the insulin receptor, thereby promoting the activation of JAK2 or the ins...
Moreno, Estefanía; Moreno-Delgado, David; Navarro, Gemma; Hoffmann, Hanne M.; Fuentes, Silvia; Rosell-Vilar, Santi; Gasperini, Paola; Rodríguez-Ruiz, Mar; Medrano, Mireia; Mallol, Josefa; Cortés, Antoni; Casadó, Vicent; Lluís, Carme; Ferré, Sergi; Ortiz, Jordi; Canela, Enric
2014-01-01
The general effects of cocaine are not well understood at the molecular level. What is known is that the dopamine D1 receptor plays an important role. Here we show that a key mechanism may be cocaine's blockade of the histamine H3 receptor-mediated inhibition of D1 receptor function. This blockade requires the σ1 receptor and occurs upon cocaine binding to σ1-D1-H3 receptor complexes. The cocaine-mediated disruption leaves an uninhibited D1 receptor that activates Gs, freely recruits β-arrestin, increases p-ERK 1/2 levels, and induces cell death when over activated. Using in vitro assays with transfected cells and in ex vivo experiments using both rats acutely treated or self-administered with cocaine along with mice depleted of σ1 receptor, we show that blockade of σ1 receptor by an antagonist restores the protective H3 receptor-mediated brake on D1 receptor signaling and prevents the cell death from elevated D1 receptor signaling. These findings suggest that a combination therapy of σ1R antagonists with H3 receptor agonists could serve to reduce some effects of cocaine. PMID:24599455
GABA(B) receptor modulation of feedforward inhibition through hippocampal neurogliaform cells.
Price, Christopher J; Scott, Ricardo; Rusakov, Dmitri A; Capogna, Marco
2008-07-02
Feedforward inhibition of neurons is a fundamental component of information flow control in the brain. We studied the roles played by neurogliaform cells (NGFCs) of stratum lacunosum moleculare of the hippocampus in providing feedforward inhibition to CA1 pyramidal cells. We recorded from synaptically coupled pairs of anatomically identified NGFCs and CA1 pyramidal cells and found that, strikingly, a single presynaptic action potential evoked a biphasic unitary IPSC (uIPSC), consisting of two distinct components mediated by GABA(A) and GABA(B) receptors. A GABA(B) receptor-mediated unitary response has not previously been observed in hippocampal excitatory neurons. The decay of the GABA(A) receptor-mediated response was slow (time constant = 50 ms), and was tightly regulated by presynaptic GABA(B) receptors. Surprisingly, the GABA(B) receptor ligands baclofen and (2S)-3-{[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl}(phenylmethyl)phosphinic acid (CGP55845), while affecting the NGFC-mediated uIPSCs, had no effect on action potential-evoked presynaptic Ca2+ signals monitored in individual axonal boutons of NGFCs with two-photon microscopy. In contrast, baclofen clearly depressed presynaptic Ca2+ transients in non-NGF interneurons. Changes in extracellular Ca2+ concentration that mimicked the effects of baclofen or CGP55845 on uIPSCs significantly altered presynaptic Ca2+ transients. Electrophysiological data suggest that GABA(B) receptors expressed by NGFCs contribute to the dynamic control of the excitatory input to CA1 pyramidal neurons from the temporoammonic path. The NGFC-CA1 pyramidal cell connection therefore provides a unique and subtle mechanism to shape the integration time domain for signals arriving via a major excitatory input to CA1 pyramidal cells.
4-Hydroxynonenal activates Src through a non-canonical pathway that involves EGFR/PTP1B
Zhang, Hongqiao; Forman, Henry Jay
2015-01-01
Src, a non-receptor protein tyrosine kinase involved in many biological processes, can be activated through both redox-dependent and independent mechanisms. 4-Hydroxy-2-nonenal (HNE) is a lipid peroxidation product that is increased in pathophysiological conditions associated with Src activation. This study examined how HNE activates human c-Src. In the canonical pathway Src activation is initiated by dephosphorylation of pTyr530 followed by conformational change that causes Src auto-phosphorylation at Tyr419 and its activation. HNE increased Src activation in both dose- and time-dependent manner, while it also increased Src phosphorylation at Tyr530 (pTyr530 Src), suggesting that HNE activated Src via a non-canonical mechanism. Protein tyrosine phosphatase 1B inhibitor (539741), at concentrations that increased basal pTyr530 Src, also increased basal Src activity and significantly reduced HNE-mediated Src activation. The EGFR inhibitor, AG1478, and EGFR silencing, abrogated HNE-mediated EGFR activation and inhibited basal and HNE-induced Src activity. In addition, AG1478 also eliminated the increase of basal Src activation by a PTP1B inhibitor. Taken together these data suggest that HNE can activate Src partly through a non-canonical pathway involving activation of EGFR and inhibition of PTP1B. PMID:26453921
PINK1 positively regulates IL-1β-mediated signaling through Tollip and IRAK1 modulation
Directory of Open Access Journals (Sweden)
Lee Hyun Jung
2012-12-01
Full Text Available Abstract Background Parkinson disease (PD is characterized by a slow, progressive degeneration of dopaminergic neurons in the substantianigra. The cause of neuronal loss in PD is not well understood, but several genetic loci, including PTEN-induced putative kinase 1 (PINK1, have been linked to early-onset autosomal recessive forms of familial PD. Neuroinflammation greatly contributes to PD neuronal degeneration and pathogenesis. IL-1 is one of the principal cytokines that regulates various immune and inflammatory responses via the activation of the transcription factors NF-κB and activating protein-1. Despite the close relationship between PD and neuroinflammation, the functional roles of PD-linked genes during inflammatory processes remain poorly understood. Methods To explore the functional roles of PINK1 in response to IL-1β stimulation, HEK293 cells, mouse embryonic fibroblasts derived from PINK1-null (PINK1−/− and control (PINK1+/+ mice, and 293 IL-1RI cells stably expressing type 1 IL-1 receptor were used. Immunoprecipitation and western blot analysis were performed to detect protein–protein interaction and protein ubiquitination. To confirm the effect of PINK1 on NF-κB activation, NF-κB-dependent firefly luciferase reporter assay was conducted. Results PINK1 specifically binds two components of the IL-1-mediated signaling cascade, Toll-interacting protein (Tollip and IL-1 receptor-associated kinase 1 (IRAK1. The association of PINK1 with Tollip, a negative regulator of IL-1β signaling, increases upon IL-1β stimulation, which then facilitates the dissociation of Tollip from IRAK1 as well as the assembly of the IRAK1–TNF receptor-associated factor 6 (TRAF6 complex. PINK1 also enhances Lys63-linked polyubiquitination of IRAK1, an essential modification of recruitment of NF-κB essential modulator and subsequent IκB kinase activation, and increases formation of the intermediate signalosome including IRAK1, TRAF6, and
αvβ5 Integrin/FAK/PGC-1α Pathway Confers Protective Effects on Retinal Pigment Epithelium.
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Murilo F Roggia
Full Text Available To elucidate the mechanism of the induction of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α by photoreceptor outer segments (POS and its effects on retinal pigment epithelium (RPE.PGC-1α upregulation by POS was confirmed in ARPE-19 cells and in RPE ex vivo. To elucidate the mechanism, siRNAs against β5 integrin, CD36, Mer tyrosine kinase (MerTK, and Atg5, blocking antibodies against CD36 and MerTK, and a specific inhibitor for focal adhesion kinase (FAK were used. We examined the effect of POS-induced PGC-1α upregulation on the levels of reactive oxygen species (ROS, mitochondrial biogenesis, senescence-associated β-galactosidase (SA-β-gal after H2O2 treatment, and lysosomal activity. Lysosomal activity was evaluated through transcriptional factor EB and its target genes, and the activity of cathepsin D. Lipid metabolism after POS treatment was assessed using Oil Red O and BODIPY C11. RPE phenotypes of PGC-1α-deficient mice were examined.POS-induced PGC-1α upregulation was suppressed by siRNA against β5 integrin and a FAK inhibitor. siRNAs and blocking antibodies against CD36 and MerTK enhanced the effect of POS on PGC-1α. The upregulation of PGC-1α increased the levels of mRNA for antioxidant enzymes and stimulated mitochondrial biogenesis, decreased ROS levels, and reduced SA-β-gal staining in H2O2-treated ARPE-19 cells. PGC-1α was critical for lysosomal activity and lipid metabolism after POS treatment. PGC-1α-deficient mice demonstrated an accumulation of type 2 lysosomes in RPE, thickening of Bruch's membrane, and poor choriocapillaris vasculature.The binding, but not the internalization of POS confers protective effects on RPE cells through the αvβ5 integrin/FAK/PGC-1α pathway.
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van Doorn Ruben
2012-06-01
Full Text Available Abstract Background The sphingosine 1-phosphate (S1P receptor modulator FTY720P (Gilenya® potently reduces relapse rate and lesion activity in the neuroinflammatory disorder multiple sclerosis. Although most of its efficacy has been shown to be related to immunosuppression through the induction of lymphopenia, it has been suggested that a number of its beneficial effects are related to altered endothelial and blood–brain barrier (BBB functionality. However, to date it remains unknown whether brain endothelial S1P receptors are involved in the maintenance of the function of the BBB thereby mediating immune quiescence of the brain. Here we demonstrate that the brain endothelial receptor S1P5 largely contributes to the maintenance of brain endothelial barrier function. Methods We analyzed the expression of S1P5 in human post-mortem tissues using immunohistochemistry. The function of S1P5 at the BBB was assessed in cultured human brain endothelial cells (ECs using agonists and lentivirus-mediated knockdown of S1P5. Subsequent analyses of different aspects of the brain EC barrier included the formation of a tight barrier, the expression of BBB proteins and markers of inflammation and monocyte transmigration. Results We show that activation of S1P5 on cultured human brain ECs by a selective agonist elicits enhanced barrier integrity and reduced transendothelial migration of monocytes in vitro. These results were corroborated by genetically silencing S1P5 in brain ECs. Interestingly, functional studies with these cells revealed that S1P5 strongly contributes to brain EC barrier function and underlies the expression of specific BBB endothelial characteristics such as tight junctions and permeability. In addition, S1P5 maintains the immunoquiescent state of brain ECs with low expression levels of leukocyte adhesion molecules and inflammatory chemokines and cytokines through lowering the activation of the transcription factor NFκB. Conclusion Our
Stress-induced decrease of uterine blood flow in sheep is mediated by alpha 1-adrenergic receptors.
Dreiling, Michelle; Bischoff, Sabine; Schiffner, Rene; Rupprecht, Sven; Kiehntopf, Michael; Schubert, Harald; Witte, Otto W; Nathanielsz, Peter W; Schwab, Matthias; Rakers, Florian
2016-09-01
Prenatal maternal stress can be transferred to the fetus via a catecholamine-dependent decrease of uterine blood flow (UBF). However, it is unclear which group of adrenergic receptors mediates this mechanism of maternal-fetal stress transfer. We hypothesized that in sheep, alpha 1-adrenergic receptors may play a key role in catecholamine mediated UBF decrease, as these receptors are mainly involved in peripheral vasoconstriction and are present in significant number in the uterine vasculature. After chronic instrumentation at 125 ± 1 days of gestation (dGA; term 150 dGA), nine pregnant sheep were exposed at 130 ± 1 dGA to acute isolation stress for one hour without visual, tactile, or auditory contact with their flockmates. UBF, blood pressure (BP), heart rate (HR), stress hormones, and blood gases were determined before and during this isolation challenge. Twenty-four hours later, experiments were repeated during alpha 1-adrenergic receptor blockage induced by a continuous intravenous infusion of urapidil. In both experiments, ewes reacted to isolation with an increase in serum norepinephrine, cortisol, BP, and HR as typical signs of activation of sympatho-adrenal and the hypothalamic-pituitary-adrenal axis. Stress-induced UBF decrease was prevented by alpha 1-adrenergic receptor blockage. We conclude that UBF decrease induced by maternal stress in sheep is mediated by alpha 1-adrenergic receptors. Future studies investigating prevention strategies of impact of prenatal maternal stress on fetal health should consider selective blockage of alpha 1-receptors to interrupt maternal-fetal stress transfer mediated by utero-placental malperfusion.
Receptor oligomerization in family B1 of G-protein-coupled receptors
DEFF Research Database (Denmark)
Roed, Sarah Norklit; Ørgaard, Anne; Jørgensen, Rasmus
2012-01-01
, the glucagon receptor, and the receptors for parathyroid hormone (PTHR1 and PTHR2). The dysregulation of several family B1 receptors is involved in diseases, such as diabetes, chronic inflammation, and osteoporosis which underlines the pathophysiological importance of this GPCR subfamily. In spite of this......, investigation of family B1 receptor oligomerization and especially its pharmacological importance is still at an early stage. Even though GPCR oligomerization is a well-established phenomenon, there is a need for more investigations providing a direct link between these interactions and receptor functionality......The superfamily of the seven transmembrane G-protein-coupled receptors (7TM/GPCRs) is the largest family of membrane-associated receptors. GPCRs are involved in the pathophysiology of numerous human diseases, and they constitute an estimated 30-40% of all drug targets. During the last two decades...
International Nuclear Information System (INIS)
O'Sullivan, Saoirse E.; Tarling, Elizabeth J.; Bennett, Andrew J.; Kendall, David A.; Randall, Michael D.
2005-01-01
Cannabinoids have widespread effects on the cardiovascular system, only some of which are mediated via G-protein-coupled cell surface receptors. The active ingredient of cannabis, Δ 9 -tetrahydrocannabinol (THC), causes acute vasorelaxation in various arteries. Here we show for the first time that THC also causes slowly developing vasorelaxation through activation of peroxisome proliferator-activated receptors gamma (PPARγ). In vitro, THC (10 μM) caused time-dependent vasorelaxation of rat isolated arteries. Time-dependent vasorelaxation to THC was similar to that produced by the PPARγ agonist rosiglitazone and was inhibited by the PPARγ antagonist GW9662 (1 μM), but not the cannabinoid CB 1 receptor antagonist AM251 (1 μM). Time-dependent vasorelaxation to THC requires an intact endothelium, nitric oxide, production of hydrogen peroxide, and de novo protein synthesis. In transactivation assays in cultured HEK293 cells, THC-activated PPARγ, transiently expressed in combination with retinoid X receptor α and a luciferase reporter gene, in a concentration-dependent manner (100 nM-10 μM). In vitro incubation with THC (1 or 10 μM, 8 days) stimulated adipocyte differentiation in cultured 3T3L1 cells, a well-accepted property of PPARγ ligands. The present results provide strong evidence that THC is a PPARγ ligand, stimulation of which causes time-dependent vasorelaxation, implying some of the pleiotropic effects of cannabis may be mediated by nuclear receptors
DEFF Research Database (Denmark)
Amrutkar, Dipak V; Ploug, Kenneth B; Hay-Schmidt, Anders
2012-01-01
Triptans, a family of 5-hydroxytryptamine (5-HT) 1B, 1D, and 1F receptor agonists, are used in the acute treatment of migraine attacks. The site of action and subtypes of the 5-HT(1) receptor that mediate the antimigraine effect have still to be identified. This study investigated the mRNA expres......Triptans, a family of 5-hydroxytryptamine (5-HT) 1B, 1D, and 1F receptor agonists, are used in the acute treatment of migraine attacks. The site of action and subtypes of the 5-HT(1) receptor that mediate the antimigraine effect have still to be identified. This study investigated the m......RNA expression of these receptors and the role of 5-HT(1) receptor subtypes in controlling the release of calcitonin gene-related peptide (CGRP) in rat dura mater, trigeminal ganglion (TG), and trigeminal nucleus caudalis (TNC). The mRNA for each receptor subtype was quantified by quantitative real...
Smith, Caroline C; McMahon, Lori L
2006-08-16
Estradiol, through activation of genomic estrogen receptors, induces changes in synaptic morphology and function in hippocampus, a brain region important for memory acquisition. Specifically, this hormone increases CA1 pyramidal cell dendritic spine density, NMDA receptor (NMDAR)-mediated transmission, and the magnitude of long-term potentiation (LTP) at CA3-CA1 synapses. We recently reported that the estradiol-induced increase in LTP magnitude occurs only when there is a simultaneous increase in the fractional contribution of NMDAR-mediated transmission relative to AMPA receptor transmission, suggesting a direct role for the increase in NMDAR transmission to the heightened LTP magnitude. Estradiol has been shown to increase expression of the NMDAR subunit NR2B, but whether this translates into an increase in function of NR2B-containing receptors remains to be determined. Here we show that not only is the estradiol-induced increase in NMDAR transmission mediated by NR2B-containing receptors, but blocking these receptors using RO25-6981 [R-(R,S)-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperidine propranol] (0.5 microM), an NR2B selective antagonist, prevents the estradiol-induced increase in LTP magnitude. Thus, our data show a causal link between the estradiol-induced increase in transmission mediated by NR2B-containing NMDARs and the increase in LTP magnitude.
International Nuclear Information System (INIS)
Huang, Chiun-Wei; Li, Zibo; Cai, Hancheng; Chen, Kai; Shahinian, Tony; Conti, Peter S.
2011-01-01
The ability of PET to aid in the diagnosis and management of recurrent and/or disseminated metastatic prostate cancer may be enhanced by the development of novel prognostic imaging probes. Accumulating experimental evidence indicates that overexpression of integrin α 2 β 1 may correlate with progression in human prostate cancer. In this study, 64 Cu-labeled integrin α 2 β 1 -targeted PET probes were designed and evaluated for the imaging of prostate cancer. DGEA peptides conjugated with a bifunctional chelator (BFC) were developed to image integrin α 2 β 1 expression with PET in a subcutaneous PC-3 xenograft model. The microPET images were reconstructed by a two-dimensional ordered subsets expectation maximum algorithm. The average radioactivity accumulation within a tumor or an organ was quantified from the multiple region of interest volumes. The PET tracer demonstrated prominent tumor uptake in the PC-3 xenograft (integrin α 2 β 1 -positive). The receptor specificity was confirmed in a blocking experiment. Moreover, the low tracer uptake in a CWR-22 tumor model (negative control) further confirmed the receptor specificity. The sarcophagine-conjugated DGEA peptide allows noninvasive imaging of tumor-associated α 2 β 1 expression, which may be a useful PET probe for evaluating the metastatic potential of prostate cancer. (orig.)
International Nuclear Information System (INIS)
Park, Catherine C.; Park, Catherine C.; Zhang, Hui J.; Yao, Evelyn S.; Park, Chong J.; Bissell, Mina J.
2008-01-01
β1 integrin signaling has been shown to mediate cellular resistance to apoptosis after exposure to ionizing radiation (IR). Other signaling molecules that increase resistance include Akt, which promotes cell survival downstream of β1 integrin signaling. We showed previously that β1 integrin inhibitory antibodies, AIIB2, enhance apoptosis and decrease growth in human breast cancer cells in 3 dimensional laminin-rich extracellular matrix (3D lrECM) cultures and in vivo. Here we asked whether AIIB2 could synergize with IR to modify Akt-mediated IR resistance. We used 3D lrECM cultures to test the optimal combination of AIIB2 with IR treatment of two breast cancer cell lines, MCF-7 and HMT3522-T4-2, as well as T4-2 myr-Akt breast cancer colonies or HMT3522-S-1, which form normal organotypic structures in 3D lrECM. Colonies were assayed for apoptosis and β1 integrin/Akt signaling pathways were evaluated using western blot. In addition, mice bearing MCF-7 xenografts were used to validate the findings in 3D lrECM. We report that AIIB2 increased apoptosis optimally post-IR by down regulating Akt in breast cancer colonies in 3D lrECM. In vivo, addition of AIIB2 after IR significantly enhanced tumor growth inhibition and apoptosis compared to either treatment alone. Remarkably, the degree of tumor growth inhibition using AIIB2 plus 2 Gy radiation was similar to that of 8 Gy alone. We showed previously that AIIB2 had no discernible toxicity in mice; here, its addition allowed for a significant reduction in the IR dose that was necessary to achieve comparable growth inhibition and apoptosis in breast cancer xenografts in vivo
Energy Technology Data Exchange (ETDEWEB)
Park, Catherine C.; Park, Catherine C.; Zhang, Hui J.; Yao, Evelyn S.; Park, Chong J.; Bissell, Mina J.
2008-06-02
{beta}1 integrin signaling has been shown to mediate cellular resistance to apoptosis after exposure to ionizing radiation (IR). Other signaling molecules that increase resistance include Akt, which promotes cell survival downstream of {beta}1 integrin signaling. We showed previously that {beta}1 integrin inhibitory antibodies, AIIB2, enhance apoptosis and decrease growth in human breast cancer cells in 3 dimensional laminin-rich extracellular matrix (3D lrECM) cultures and in vivo. Here we asked whether AIIB2 could synergize with IR to modify Akt-mediated IR resistance. We used 3D lrECM cultures to test the optimal combination of AIIB2 with IR treatment of two breast cancer cell lines, MCF-7 and HMT3522-T4-2, as well as T4-2 myr-Akt breast cancer colonies or HMT3522-S-1, which form normal organotypic structures in 3D lrECM. Colonies were assayed for apoptosis and {beta}1 integrin/Akt signaling pathways were evaluated using western blot. In addition, mice bearing MCF-7 xenografts were used to validate the findings in 3D lrECM. We report that AIIB2 increased apoptosis optimally post-IR by down regulating Akt in breast cancer colonies in 3D lrECM. In vivo, addition of AIIB2 after IR significantly enhanced tumor growth inhibition and apoptosis compared to either treatment alone. Remarkably, the degree of tumor growth inhibition using AIIB2 plus 2 Gy radiation was similar to that of 8 Gy alone. We showed previously that AIIB2 had no discernible toxicity in mice; here, its addition allowed for a significant reduction in the IR dose that was necessary to achieve comparable growth inhibition and apoptosis in breast cancer xenografts in vivo.
Jiang, Youde; Zhang, Qiuhua; Ye, Eun-Ah
2014-01-01
Purpose Determine whether Compound 49b treatment ameliorates retinal changes due to the lack of β2-adrenergic receptor signaling. Methods Using retinas from 3-month-old β2-adrenergic receptor-deficient mice, we treated mice with our novel β1-/β2-adrenergic receptor agonist, Compound 49b, to assess the effects of adrenergic agonists acting only on β1-adrenergic receptors due to the absence of β2-adrenergic receptors. Western blotting or enzyme-linked immunosorbent assay (ELISA) analyses were performed for β1- and β2-adrenergic receptors, as well as key insulin resistance proteins, including TNF-α, SOCS3, IRS-1Ser307, and IRTyr960. Analyses were also performed on key anti- and proapoptotic proteins: Akt, Bcl-xL, Bax, and caspase 3. Electroretinogram analyses were conducted to assess functional changes, while histological assessment was conducted for changes in retinal thickness. Results A 2-month treatment of β2-adrenergic receptor-deficient mice with daily eye drops of 1 mM Compound 49b, a novel β1- and β2-adrenergic receptor agonist, reversed the changes in insulin resistance markers (TNF-α and SOCS3) observed in untreated β2-adrenergic receptor-deficient mice, and concomitantly increased morphological integrity (retinal thickness) and functional responses (electroretinogram amplitude). These results suggest that stimulating β1-adrenergic receptors on retinal endothelial cells or Müller cells can compensate for the loss of β2-adrenergic receptor signaling on Müller cells, restore insulin signal transduction, reduce retinal apoptosis, and enhance retinal function. Conclusions Since our previous studies with β1-adrenergic receptor knockout mice confirmed that the reverse also occurs (β2-adrenergic receptor stimulation can compensate for the loss of β1-adrenergic receptor activity), it appears that increased activity in either of these pathways alone is sufficient to block insulin resistance–based retinal cell apoptosis. PMID:24966659
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Weijuan Li
2014-01-01
Full Text Available Our study intended to prove whether agonistic autoantibodies to angiotensin II type 1 receptor (AT1-AAs exist in patients with coronary heart disease (CHD and affect the human endothelial cell (HEC by upregulating proinflammatory cytokines expression involved in NF-κB pathway. Antibodies were determined by chronotropic responses of cultured neonatal rat cardiomyocytes coupled with receptor-specific antagonists (valsartan and AT1-EC2 as described previously. Interleukin-6 (IL-6, vascular cell adhesion molecule-1 (VCAM-1, and monocyte chemotactic protein-1 (MCP-1 expression were improved at both mRNA and protein levels in HEC, while NF-κB in the DNA level was improved detected by electrophoretic mobility shift assays (EMSA. These improvements could be inhibited by specific AT1 receptor blocker valsartan, NF-κB blocker pyrrolidine dithiocarbamate (PDTC, and specific short peptides from the second extracellular loop of AT1 receptor. These results suggested that AT1-AAs, via the AT1 receptor, induce expression of proinflammatory cytokines involved in the activation of NF-κB. AT1-AAs may play a great role in the pathogenesis of the acute coronary syndrome by mediating vascular inflammatory effects involved in the NF-κB pathway.
Chao, Honglu; Liu, Yinlong; Lin, Chao; Xu, Xiupeng; Li, Zheng; Bao, Zhongyuan; Fan, Liang; Tao, Chao; Zhao, Lin; Liu, Yan; Wang, Xiaoming; You, Yongping; Liu, Ning; Ji, Jing
2018-06-09
Phospholipase A 2 is a known aggravator of inflammation and deteriorates neurological outcomes after traumatic brain injury (TBI), however the exact inflammatory mechanisms remain unknown. This study investigated the role of bradykinin and its receptor, which are known initial mediators within inflammation activation, as well as the mechanisms of the cytosolic phospholipase A 2 (cPLA 2 )-related inflammatory responses after TBI. We found that cPLA 2 and bradykinin B2 receptor were upregulated after a TBI. Rats treated with the bradykinin B2 receptor inhibitor LF 16-0687 exhibited significantly less cPLA 2 expression and related inflammatory responses in the brain cortex after sustaining a controlled cortical impact (CCI) injury. Both the cPLA 2 inhibitor and the LF16-0687 improved CCI rat outcomes by decreasing neuron death and reducing brain edema. The following TBI model utilized both primary astrocytes and primary neurons in order to gain further understanding of the inflammation mechanisms of the B2 bradykinin receptor and the cPLA 2 in the central nervous system. There was a stronger reaction from the astrocytes as well as a protective effect of LF16-0687 after the stretch injury and bradykinin treatment. The protein kinase C pathway was thought to be involved in the B2 bradykinin receptor as well as the cPLA 2 -related inflammatory responses. Rottlerin, a Protein Kinase C (PKC) δ inhibitor, decreased the activity of the cPLA 2 activity post-injury, and LF16-0687 suppressed both the PKC pathway and the cPLA 2 activity within the astrocytes. These results indicated that the bradykinin B2 receptor-mediated pathway is involved in the cPLA 2 -related inflammatory response from the PKC pathway. Copyright © 2018. Published by Elsevier B.V.
Sahler, Julie; Woeller, Collynn F; Phipps, Richard P
2014-01-01
Circulating blood microparticles are submicron vesicles released primarily by megakaryocytes and platelets that act as transcellular communicators. Inflammatory conditions exhibit elevated blood microparticle numbers compared to healthy conditions. Direct functional consequences of microparticle composition, especially internal composition, on recipient cells are poorly understood. Our objective was to evaluate if microparticle composition could impact the function of recipient cells, particularly during inflammatory provocation. We therefore engineered the composition of megakaryocyte culture-derived microparticles to generate distinct microparticle populations that were given to human monocytes to assay for influences recipient cell function. Herein, we tested the responses of monocytes exposed to either control microparticles or microparticles that contain the anti-inflammatory transcription factor, peroxisome proliferator-activated receptor-γ (PPARγ). In order to normalize relative microparticle abundance from two microparticle populations, we implemented a novel approach that utilizes a Nanodrop Spectrophotometer to assay for microparticle density rather than concentration. We found that when given to peripheral blood mononuclear cells, microparticles were preferentially internalized by CD11b+ cells, and furthermore, microparticle composition had a profound functional impact on recipient monocytes. Specifically, microparticles containing PPARγ reduced activated monocyte production of the proinflammatory cytokines interleukin-8 and monocyte chemotactic protein-1 compared to activated monocytes exposed to control microparticles. Additionally, treatment with PPARγ microparticles greatly increased monocyte cell adherence. This change in morphology occurred simultaneously with increased production of the key extracellular matrix protein, fibronectin and increased expression of the fibronectin-binding integrin, ITGA5. PPARγ microparticles also changed monocyte
Kwok, Hoi-Hin
2017-05-18
MicroRNAs (miRNAs) are a family of non-coding RNAs that play crucial roles in regulating various normal cellular responses. Recent studies revealed that the canonical miRNA biogenesis pathway is subject to sophisticated regulation. Hormonal control of miRNA biogenesis by androgen and estrogen has been demonstrated, but the direct effects of the glucocorticoid receptor (GR) on miRNA biogenesis are unknown. This study revealed the role of GR in miRNA maturation. We showed that two GR agonists, dexamethasone and ginsenoside-Rg1 rapidly suppressed the expression of mature miR-15b, miR-23a, and miR-214 in human endothelial cells. RNA pulldown coupled with proteomic analysis identified GTPase-activating protein (SH3 domain) binding protein 1 (G3BP1) as one of the RNA-binding proteins mediating GR-regulated miRNA maturation. Activated GR induced phosphorylation of v-AKT Murine Thymoma Viral Oncogene Homologue (AKT) kinase, which in turn phosphorylated and promoted nuclear translocation of G3BP1. The nuclear G3BP1 bound to the G3BP1 consensus sequence located on primary miR-15b~16-2 and miR-23a~27a~24-2 to inhibit their maturation. The findings from this study have advanced our understanding of the non-genomic effects of GR in the vascular system.
Endothelial adhesion molecules and leukocyte integrins in preeclamptic patients.
Haller, H; Ziegler, E M; Homuth, V; Drab, M; Eichhorn, J; Nagy, Z; Busjahn, A; Vetter, K; Luft, F C
1997-01-01
Endothelial cell activation is important in the pathogenesis of preeclampsia; however, the nature of the activation is unknown. We investigated 22 patients with preeclampsia. 29 normotensive pregnancies, and 18 nonpregnant women to test the hypothesis that serum from preeclamptic patients induces expression of intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) and stimulates intracellular free calcium concentrations [Ca2+]i in cultured endothelial cells. We then asked whether the corresponding integrin adhesive counter receptors lymphocyte function-associated antigen-1 (CD11a/CD18), macrophage-1 antigen (CD11b/CD18), p150,95 (CD11c/CD18), and very late activation antigen-4 (CD49/CD29) are increased in patients with preeclampsia. In the pregnant women, the measurements were conducted both before and after delivery. Integrin expression was measured by fluorescent antibody cell sorting analysis using monoclonal antibodies. ICAM-1 and VCAM-1 were analyzed on endothelial cells by enzyme-linked immunosorbent assay. [Ca2+]i was measured with fura 2. Serum from preeclamptic patients increased endothelial cell ICAM-1 expression but not VCAM-1 expression. Preeclamptic patients' serum also increased [Ca2+]i in endothelial cells compared with serum from normal nonpregnant or normal pregnant women. Endothelial cell [Ca2+]i concentrations were correlated with the ICAM-1 expression in preeclamptic patients (r = .80, P preclampsia and normal pregnancy compared with the nonpregnant state. The expression decreased significantly after delivery in both groups. Our results demonstrate that serum from preeclamptic women induces increased ICAM-1 surface expression on endothelial cells, while the expression of the integrin counterreceptors was not different. The effect on endothelial cells may be related to an increase in [Ca2+]i. The effect on cultured endothelial cells and the rapid decrease after delivery suggests the presence of a circulating serum
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Bangfu Zhu
2016-11-01
Full Text Available The guided migration of neural cells is essential for repair in the central nervous system (CNS. Oligodendrocyte progenitor cells (OPCs will normally migrate towards an injury site to re-sheath demyelinated axons; however the mechanisms underlying this process are not well understood. Endogenous electric fields (EFs are known to influence cell migration in vivo, and have been utilised in this study to direct the migration of OPCs isolated from neonatal Sprague-Dawley rats. The OPCs were exposed to physiological levels of electrical stimulation, and displayed a marked electrotactic response that was dependent on β1 integrin, one of the key subunits of integrin receptors. We also observed that F-actin, an important component of the cytoskeleton, was re-distributed towards the leading edge of the migrating cells, and that this asymmetric rearrangement was associated with β1 integrin function.
A role for the high-density lipoprotein receptor SR-B1 in synovial inflammation via serum amyloid-A.
LENUS (Irish Health Repository)
Mullan, Ronan Hugh
2012-02-01
Acute phase apoprotein Serum Amyloid A (A-SAA), which is strongly expressed in rheumatoid arthritis synovial membrane (RA SM), induces angiogenesis, adhesion molecule expression, and matrix metalloproteinase production through the G-coupled receptor FPRL-1. Here we report alternative signaling through the high-density lipoprotein receptor scavenger receptor-class B type 1 (SR-B1). Quantitative expression\\/localization of SR-B1 in RA SM, RA fibroblast-like cells (FLCs), and microvascular endothelial cells (ECs) was assessed by Western blotting and immunohistology\\/fluorescence. A-SAA-mediated effects were examined using a specific antibody against SR-B1 or amphipathic alpha-Helical Peptides (the SR-B1 antagonists L-37pA and D-37pA), in RA FLCs and ECs. Adhesion molecule expression and cytokine production were quantified using flow cytometry and ELISA. SR-B1 was strongly expressed in the RA SM lining layer and endothelial\\/perivascular regions compared with osteoarthritis SM or normal control synovium. Differential SR-B1 expression in RA FLC lines (n = 5) and ECs correlated closely with A-SAA, but not tumor necrosis factor alpha-induced intercellular adhesion molecule-1 upregulation. A-SAA-induced interleukin-6 and -8 production was inhibited in the presence of anti-SR-B1 in human microvascular endothelial cells and RA FLCs. Moreover, D-37pA and L-37pA inhibited A-SAA-induced vascular cell adhesion molecule-1 and intercellular adhesion molecule expression from ECs in a dose-dependent manner. As SR-B1 is expressed in RA synovial tissue and mediates A-SAA-induced pro-inflammatory pathways, a better understanding of A-SAA-mediated inflammatory pathways may lead to novel treatment strategies for RA.
Cattavarayane, Sandhanakrishnan; Palovuori, Riitta; Tanjore Ramanathan, Jayendrakishore; Manninen, Aki
2015-02-27
The growth properties and self-renewal capacity of embryonic stem (ES) cells are regulated by their immediate microenvironment such as the extracellular matrix (ECM). Integrins, a central family of cellular ECM receptors, have been implicated in these processes but their specific role in ES cell self-renewal remains unclear. Here we have studied the effects of different ECM substrates and integrins in mouse ES cells in the absence of Leukemia Inhibitory Factor (LIF) using short-term assays as well as long-term cultures. Removal of LIF from ES cell culture medium induced morphological differentiation of ES cells into polarized epistem cell-like cells. These cells maintained epithelial morphology and expression of key stemness markers for at least 10 passages in the absence of LIF when cultured on laminin, fibronectin or collagen IV substrates. The specific functional roles of α6-, αV- and β1-integrin subunits were dissected using stable lentivirus-mediated RNAi methodology. β1-integrins were required for ES cell survival in long-term cultures and for the maintenance of stem cell marker expression. Inhibition of α6-integrin expression compromised self-renewal on collagen while αV-integrins were required for robust ES cell adhesion on laminin. Analysis of the stemness marker expression revealed subtle differences between α6- and αV-depleted ES cells but the expression of both was required for optimal self-renewal in long-term ES cell cultures. In the absence of LIF, long-term ES cell cultures adapt an epistem cell-like epithelial phenotype and retain the expression of multiple stem cell markers. Long-term maintenance of such self-renewing cultures depends on the expression of β1-, α6- and αV-integrins.
DEFF Research Database (Denmark)
Eskesen, Karen; Edvinsson, Lars
2006-01-01
The aim of this study was to examine if endothelin ET(B) receptor-mediated contraction occurred in isolated segments of rat coronary arteries during organ culture. Presence of contractile endothelin ET(B) receptors was studied by measuring the change in isometric tension in rings of left anterior......(+)-solution was not modified after 1 day in culture medium. The experiments indicate that organ culture of rat coronary arteries upregulate endothelin ET(B) receptor-mediated contraction by inducing synthesis of new protein....... descending coronary arteries isolated from hearts of rats as response to application of the selective endothelin ET(B) receptor agonist, Sarafotoxin 6c and endothelin-1. In segments cultured 1 day in serum free Dulbecco's Modified Eagle's Medium, Sarafotoxin 6c induced a concentration dependent contraction...
Versleijen, Michelle W J; van Esterik, Joantine C J; Roelofs, Hennie M J; van Emst-de Vries, Sjenet E; Willems, Peter H G M; Wanten, Geert J A
2009-02-01
Lipid-induced immune modulation might contribute to the increased infection rate that is observed in patients using parenteral nutrition. We previously showed that emulsions containing medium-chain triglycerides (LCT/MCTs or pure MCTs), but not pure long-chain triglycerides (LCTs), impair neutrophil functions, modulate cell-signaling and induce neutrophil activation in vitro. It has recently been shown that medium-chain fatty acids are ligands for GPR84, a pertussis toxin (PT)-sensitive G-protein-coupled receptor (GPCR). This finding urged us to investigate whether MCT-induced neutrophil activation is mediated by PT-sensitive GPCRs. Neutrophils isolated from blood of healthy volunteers were pre-incubated with PT (0.5-1 microg/mL, 1.5 h) and analyzed for the effect of this pre-incubation on LCT/MCT (2.5 mmol/L)-dependent modulation of serum-treated zymosan (STZ)-induced intracellular Ca(2+) mobilization and on LCT/MCT (5 mmol/L)-induced expression of cell surface adhesion (CD11b) and degranulation (CD66b) markers and oxygen radical (ROS) production. PT did not inhibit the effects of LCT/MCT on the STZ-induced increase in cytosolic free Ca(2+) concentration. LCT/MCT increased ROS production to 146% of unstimulated cells. However, pre-incubation with PT did not inhibit the LCT/MCT-induced ROS production. Furthermore, the LCT/MCT-induced increase in CD11b and CD66b expression (196% and 235% of unstimulated cells, respectively) was not inhibited by pre-incubation with PT. LCT/MCT-induced neutrophil activation does not involve the action of a PT-sensitive G-protein-coupled receptor.
International Nuclear Information System (INIS)
Sheng, Zhi-Guo; Tang, Yuan; Liu, Yu-Xiang; Yuan, Ye; Zhao, Bao-Quan; Chao, Xi-Juan; Zhu, Ben-Zhan
2012-01-01
Bisphenol (BPA) is one of the highest-volume chemicals produced worldwide, and human exposure to BPA is thought to be ubiquitous. Various rodent and in vitro studies have shown that thyroid hormone (TH) function can be impaired by BPA. However, it is still unknown if low concentrations of BPA can suppress the thyroid hormone receptor (TR) transcription. The present study aims to investigate the possible suppressing effects of low concentrations of BPA on TR transcription and the involved mechanism(s) in CV-1 cells derived from cercopithecus aethiops monkey kidneys. Using gene reporter assays, BPA at concentrations as low as 10 −9 M suppresses TR or steroid receptor coactivator-1(SRC-1)-enhanced TR transcription, but not reducing TR/SRC-1 interaction in mammalian two-hybrid and glutathione S-transferase pull-down studies. It has been further shown that both nuclear receptor co-repressor (N-CoR) and silencing mediator for retinoid and thyroid hormone receptors (SMRT) are recruited to the TR-β1 by BPA in the presence of physiologic concentrations of T3 or T4. However, the overexpression of β3 integrin or c-Src significantly reduces BPA-induced recruitment of N-CoR/SMRT to TR or suppression of TR transcription. Furthermore, BPA inhibits the T3/T4-mediated interassociation of the β3 integrin/c-Src/MAPK/TR-β1 pathways by the co-immunoprecipitation. These results indicate that low concentrations of BPA suppress the TR transcription by disrupting physiologic concentrations of T3/T4-mediated β3 integrin/c-Src/MAPK/TR-β1 pathways, followed by recruiting N-CoR/SMRT to TR-β1, providing a novel insight regarding the TH disruption effects of low concentration BPA. -- Highlights: ► Environmentally relevant concentrations of BPA suppress TR transcription. ► BPA recruits the N-CoR/SMRT to TR under the physiologic concentrations of T3/T4. ► BPA disrupts T3/T4-mediated β3 integrin/c-Src/MAPK/TR-β1 pathways.
Energy Technology Data Exchange (ETDEWEB)
Sheng, Zhi-Guo [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Science, Chinese Academy of Sciences, 18 Shuangqing Road, Beijing 100085 (China); Tang, Yuan [Department of Pharmaceutics, College of Pharmacy, Third Military Medical University, 30 Yanzheng Street, Chongqing 400038 (China); Liu, Yu-Xiang [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Science, Chinese Academy of Sciences, 18 Shuangqing Road, Beijing 100085 (China); Yuan, Ye; Zhao, Bao-Quan [Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Beijing 100850 (China); Chao, Xi-Juan [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Science, Chinese Academy of Sciences, 18 Shuangqing Road, Beijing 100085 (China); Zhu, Ben-Zhan, E-mail: bzhu@rcees.ac.cn [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Science, Chinese Academy of Sciences, 18 Shuangqing Road, Beijing 100085 (China)
2012-02-15
Bisphenol (BPA) is one of the highest-volume chemicals produced worldwide, and human exposure to BPA is thought to be ubiquitous. Various rodent and in vitro studies have shown that thyroid hormone (TH) function can be impaired by BPA. However, it is still unknown if low concentrations of BPA can suppress the thyroid hormone receptor (TR) transcription. The present study aims to investigate the possible suppressing effects of low concentrations of BPA on TR transcription and the involved mechanism(s) in CV-1 cells derived from cercopithecus aethiops monkey kidneys. Using gene reporter assays, BPA at concentrations as low as 10{sup −9} M suppresses TR or steroid receptor coactivator-1(SRC-1)-enhanced TR transcription, but not reducing TR/SRC-1 interaction in mammalian two-hybrid and glutathione S-transferase pull-down studies. It has been further shown that both nuclear receptor co-repressor (N-CoR) and silencing mediator for retinoid and thyroid hormone receptors (SMRT) are recruited to the TR-β1 by BPA in the presence of physiologic concentrations of T3 or T4. However, the overexpression of β3 integrin or c-Src significantly reduces BPA-induced recruitment of N-CoR/SMRT to TR or suppression of TR transcription. Furthermore, BPA inhibits the T3/T4-mediated interassociation of the β3 integrin/c-Src/MAPK/TR-β1 pathways by the co-immunoprecipitation. These results indicate that low concentrations of BPA suppress the TR transcription by disrupting physiologic concentrations of T3/T4-mediated β3 integrin/c-Src/MAPK/TR-β1 pathways, followed by recruiting N-CoR/SMRT to TR-β1, providing a novel insight regarding the TH disruption effects of low concentration BPA. -- Highlights: ► Environmentally relevant concentrations of BPA suppress TR transcription. ► BPA recruits the N-CoR/SMRT to TR under the physiologic concentrations of T3/T4. ► BPA disrupts T3/T4-mediated β3 integrin/c-Src/MAPK/TR-β1 pathways.
Pachathundikandi, Suneesh Kumar; Tegtmeyer, Nicole; Backert, Steffen
2013-01-01
Helicobacter pylori infections can induce pathologies ranging from chronic gastritis, peptic ulceration to gastric cancer. Bacterial isolates harbor numerous well-known adhesins, vacuolating cytotoxin VacA, protease HtrA, urease, peptidoglycan, and type IV secretion systems (T4SS). It appears that H. pylori targets more than 40 known host protein receptors on epithelial or immune cells. A series of T4SS components such as CagL, CagI, CagY, and CagA can bind to the integrin α5β1 receptor. Other targeted membrane-based receptors include the integrins αvβ3, αvβ5, and β2 (CD18), RPTP-α/β, GP130, E-cadherin, fibronectin, laminin, CD46, CD74, ICAM1/LFA1, T-cell receptor, Toll-like receptors, and receptor tyrosine kinases EGFR, ErbB2, ErbB3, and c-Met. In addition, H. pylori is able to activate the intracellular receptors NOD1, NOD2, and NLRP3 with important roles in innate immunity. Here we review the interplay of various bacterial factors with host protein receptors. The contribution of these interactions to signal transduction and pathogenesis is discussed. PMID:24280762
Directory of Open Access Journals (Sweden)
Carbone Maria
2006-09-01
Full Text Available Abstract Background Mammalian sperm-oocyte interaction at fertilization involves several combined interactions between integrins on the oocyte and integrin ligands (disintegrins on the sperm. Recent research has indicated the ability of peptides containing the RGD sequence that characterized several sperm disintegrins, to induce intracellular Ca2+ transients and to initiate parthenogenetic development in amphibian and bovine oocytes. In the present study, we investigate the hypothesis that an integrin-associated signalling may participate in oocyte activation signalling by determining the ability of a cyclic RGD-containing peptide to stimulate the activation of protein kinase C (PKC and the exocytosis of cortical granules in mouse oocytes. Methods An In-Vitro-Fertilization assay (IVF was carried in order to test the condition under which a peptide containing the RGD sequence, cyclo(Arg-Gly-Asp-D-Phe-Val, was able to inhibit sperm fusion with zona-free mouse oocytes at metaphase II stage. PKC activity was determined by means of an assay based on the ability of cell lysates to phosphorylate MARKS peptide, a specific PKC substrate. Loss of cortical granules was evaluated by measuring density in the oocyte cortex of cortical granules stained with LCA-biotin/Texas red-streptavidin. In all the experiments, effects of a control peptide containing a non RGD sequence, cyclo(Arg-Ala-Asp-D-Phe-Val, were evaluated. Results The IVF assay revealed that the fusion rate declined significantly when insemination was carried out in the presence of cyclic RGD peptide at concentrations > or = 250 microM (P Conclusion The presents results provide evidence that a cyclic RGD peptide highly effective in inhibiting sperm-oocyte interaction stimulates in mouse oocytes the activation of PKC and the exocytosis of cortical granules. These data support the view that RGD-binding receptors may function as signalling receptors giving rise integrated signalling not sufficient for
2015-10-01
H. Wagner, K. Takeda, and S. Akira. 2000 . A Toll-like receptor recognizes bacterial DNA. Nature 408: 740–745. 2. Kawai, T., and S. Akira. 2010. The...I. M. Carr , J. C. Fuller, R. M. Jackson, T. Lamb, T. A. Briggs, et al. 2009. Mutations involved in Aicardi-Goutières syndrome implicate SAMHD1 as...Moreover, recent studies from Riley and colleagues (27) suggest that ABCs negatively influence B-lineage commitment or development of bone marrow
Lifescience Database Archive (English)
Full Text Available 15662540 Modulation of Toll-interleukin 1 receptor mediated signaling. Li X, Qin J.... J Mol Med. 2005 Apr;83(4):258-66. Epub 2005 Jan 21. (.png) (.svg) (.html) (.csml) Show Modulation of Toll-i...nterleukin 1 receptor mediated signaling. PubmedID 15662540 Title Modulation of Toll-interleukin 1 receptor
Du, C; Xu, Y; Yang, K; Chen, S; Wang, X; Wang, S; Wang, C; Shen, M; Chen, F; Chen, M; Zeng, D; Li, F; Wang, T; Wang, F; Zhao, J; Ai, G; Cheng, T; Su, Y; Wang, J
2017-04-01
Estrogen is reported to be involved in thrombopoiesis and the disruption of its signaling may cause myeloproliferative disease, yet the underlying mechanisms remain largely unknown. GATA-binding factor 1 (GATA1) is a key regulator of megakaryocyte (MK) differentiation and its deficiency will lead to megakaryoblastic leukemia. Here we show that estrogen can dose-dependently promote MK polyploidization and maturation via activation of estrogen receptor beta (ERβ), accompanied by a significant upregulation of GATA1. Chromatin immunoprecipitation and a dual luciferase assay demonstrate that ERβ can directly bind the promoter region of GATA1 and activate its transcription. Steroid receptor coactivator 3 (SRC3) is involved in ERβ-mediated GATA1 transcription. The deficiency of ERβ or SRC3, similar to the inhibition of GATA1, leads to the impediment of estrogen-induced MK polyploidization and platelet production. Further investigations reveal that signal transducer and activator of transcription 1 signaling pathway downstream of GATA1 has a crucial role in estrogen-induced MK polyploidization, and ERβ-mediated GATA1 upregulation subsequently enhances nuclear factor erythroid-derived 2 expression, thereby promoting proplatelet formation and platelet release. Our study provides a deep insight into the molecular mechanisms of estrogen signaling in regulating thrombopoiesis and the pathogenesis of ER deficiency-related leukemia.
Zhao, Li-Hua; Yin, Yanting; Yang, Dehua; Liu, Bo; Hou, Li; Wang, Xiaoxi; Pal, Kuntal; Jiang, Yi; Feng, Yang; Cai, Xiaoqing; Dai, Antao; Liu, Mingyao; Wang, Ming-Wei; Melcher, Karsten; Xu, H Eric
2016-07-15
G protein-coupled receptors (GPCRs) from the secretin-like (class B) family are key players in hormonal homeostasis and are important drug targets for the treatment of metabolic disorders and neuronal diseases. They consist of a large N-terminal extracellular domain (ECD) and a transmembrane domain (TMD) with the GPCR signature of seven transmembrane helices. Class B GPCRs are activated by peptide hormones with their C termini bound to the receptor ECD and their N termini bound to the TMD. It is thought that the ECD functions as an affinity trap to bind and localize the hormone to the receptor. This in turn would allow the hormone N terminus to insert into the TMD and induce conformational changes of the TMD to activate downstream signaling. In contrast to this prevailing model, we demonstrate that human class B GPCRs vary widely in their requirement of the ECD for activation. In one group, represented by corticotrophin-releasing factor receptor 1 (CRF1R), parathyroid hormone receptor (PTH1R), and pituitary adenylate cyclase activating polypeptide type 1 receptor (PAC1R), the ECD requirement for high affinity hormone binding can be bypassed by induced proximity and mass action effects, whereas in the other group, represented by glucagon receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R), the ECD is required for signaling even when the hormone is covalently linked to the TMD. Furthermore, the activation of GLP-1R by small molecules that interact with the intracellular side of the receptor is dependent on the presence of its ECD, suggesting a direct role of the ECD in GLP-1R activation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
Petrie, Timothy A; Capadona, Jeffrey R; Reyes, Catherine D; García, Andrés J
2006-11-01
Biomimetic strategies focusing on presenting short bioadhesive oligopeptides, including the arginine-glycine-aspartic acid (RGD) motif present in numerous adhesive proteins, on a non-fouling support have emerged as promising approaches to improve cellular activities and healing responses. Nevertheless, these bio-inspired strategies are limited by low activity of the oligopeptides compared to the native ligand due to the absence of complementary or modulatory domains. In the present analysis, we generated well-defined biointerfaces presenting RGD-based ligands of increasing complexity to directly compare their biological activities in terms of cell adhesion strength, integrin binding and signaling. Mixed self-assembled monolayers of alkanethiols on gold were optimized to engineer robust supports that present anchoring groups for ligand tethering within a non-fouling, protein adsorption-resistant background. Controlled bioadhesive interfaces were generated by tethering adhesive ligands via standard peptide chemistry. On a molar basis, biointerfaces functionalized with the FNIII7-10 recombinant fragment presenting the RGD and PHSRN adhesive motifs in the correct structural context exhibited significantly higher adhesion strength, FAK activation, and cell proliferation rate than supports presenting RGD ligand or RGD-PHSRN, an oligopeptide presenting these two sites separated by a polyglycine linker. Moreover, FNIII7-10-functionalized surfaces displayed specificity for alpha5beta1 integrin, while cell adhesion to supports presenting RGD or RGD-PHSRN was primarily mediated by alphavbeta3 integrin. These results are significant to the rational engineering of bioactive materials that convey integrin binding specificity for directed cellular and tissue responses in biomedical and biotechnological applications.
Directory of Open Access Journals (Sweden)
Teresa Y. Jackson
2010-12-01
Full Text Available The vascular myogenic response is characterized by arterial constriction in response to an increase in intraluminal pressure and dilatation to a decrease in pressure. This mechanism is important for the regulation of blood flow, capillary pressure and arterial pressure. The identity of the mechanosensory mechanism(s for this response is incompletely understood but has been shown to include the integrins as cell-extracellular matrix receptors. The possibility that a cell-cell adhesion receptor is involved has not been studied. Thus, we tested the hypothesis that N-cadherin, a cell-cell adhesion molecule in vascular smooth muscle cells (VSMCs, was important for myogenic responsiveness. The purpose of this study was to investigate:
1. whether cadherin inhibition blocks myogenic responses to increases in intraluminal pressure and 2. the effect of the cadherin or integrin blockade on pressure-induced changes in [Ca2+]i. Cadherin blockade was tested in isolated rat cremaster arterioles on myogenic responses to acute pressure steps from 60 – 100 mmHg and changes in VSMC Ca2+ were measured using fura-2. In the presence of a synthetic cadherin inhibitory peptide or a function blocking antibody, myogenic responses were inhibited. In contrast, during N-cadherin blockade, pressure-induced changes in [Ca2+]i were not altered. Similarly, vessels treated with function-blocking β1- or β3-integrin antibodies maintained pressure-induced [Ca2+]i responses despite inhibition of myogenic constriction. Collectively, these data suggest that both cadherins and integrins play a fundamental role in mediating myogenic constriction but argue against their direct involvement in mediating pressure-induced [Ca2+]i increases.
de Graaf, Chris; Donnelly, Dan; Wootten, Denise; Lau, Jesper; Sexton, Patrick M.; Miller, Laurence J.; Ahn, Jung-Mo; Liao, Jiayu; Fletcher, Madeleine M.; Brown, Alastair J. H.; Zhou, Caihong; Deng, Jiejie; Wang, Ming-Wei
2016-01-01
The glucagon-like peptide (GLP)-1 receptor (GLP-1R) is a class B G protein–coupled receptor (GPCR) that mediates the action of GLP-1, a peptide hormone secreted from three major tissues in humans, enteroendocrine L cells in the distal intestine, α cells in the pancreas, and the central nervous system, which exerts important actions useful in the management of type 2 diabetes mellitus and obesity, including glucose homeostasis and regulation of gastric motility and food intake. Peptidic analogs of GLP-1 have been successfully developed with enhanced bioavailability and pharmacological activity. Physiologic and biochemical studies with truncated, chimeric, and mutated peptides and GLP-1R variants, together with ligand-bound crystal structures of the extracellular domain and the first three-dimensional structures of the 7-helical transmembrane domain of class B GPCRs, have provided the basis for a two-domain–binding mechanism of GLP-1 with its cognate receptor. Although efforts in discovering therapeutically viable nonpeptidic GLP-1R agonists have been hampered, small-molecule modulators offer complementary chemical tools to peptide analogs to investigate ligand-directed biased cellular signaling of GLP-1R. The integrated pharmacological and structural information of different GLP-1 analogs and homologous receptors give new insights into the molecular determinants of GLP-1R ligand selectivity and functional activity, thereby providing novel opportunities in the design and development of more efficacious agents to treat metabolic disorders. PMID:27630114
Cherian, Milu T; Lin, Wenwei; Wu, Jing
2015-01-01
Constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are xenobiotic sensors that enhance the detoxification and elimination of xenobiotics and endobiotics by modulating the expression of genes encoding drug-metabolizing enzymes and transporters. Elevated levels of drug-metabolizing enzymes and efflux transporters, resulting from CAR activation in various cancers, promote the elimination of chemotherapeutic agents, leading to reduced therapeutic effectiveness and acquired drug resistance. CAR inhibitors, in combination with existing chemotherapeutics, could therefore be used to attenuate multidrug resistance in cancers. Interestingly, all previously reported CAR inverse-agonists are also activators of PXR, rendering them mechanistically counterproductive in tissues where both these xenobiotic receptors are present and active. We used a directed high-throughput screening approach, followed by subsequent mechanistic studies, to identify novel, potent, and specific small-molecule CAR inhibitors that do not activate PXR. We describe here one such inhibitor, CINPA1 (CAR inhibitor not PXR activator 1), capable of reducing CAR-mediated transcription with an IC50 of ∼70 nM. CINPA1 1) is a specific xenobiotic receptor inhibitor and has no cytotoxic effects up to 30 µM; 2) inhibits CAR-mediated gene expression in primary human hepatocytes, where CAR is endogenously expressed; 3) does not alter the protein levels or subcellular localization of CAR; 4) increases corepressor and reduces coactivator interaction with the CAR ligand-binding domain in mammalian two-hybrid assays; and 5) disrupts CAR binding to the promoter regions of target genes in chromatin immunoprecipitation assays. CINPA1 could be used as a novel molecular tool for understanding CAR function. PMID:25762023
Lymphocytes Negatively Regulate NK Cell Activity via Qa-1b following Viral Infection
Directory of Open Access Journals (Sweden)
Haifeng C. Xu
2017-11-01
Full Text Available NK cells can reduce anti-viral T cell immunity during chronic viral infections, including infection with the lymphocytic choriomeningitis virus (LCMV. However, regulating factors that maintain the equilibrium between productive T cell and NK cell immunity are poorly understood. Here, we show that a large viral load resulted in inhibition of NK cell activation, which correlated with increased expression of Qa-1b, a ligand for inhibitory NK cell receptors. Qa-1b was predominantly upregulated on B cells following LCMV infection, and this upregulation was dependent on type I interferons. Absence of Qa-1b resulted in increased NK cell-mediated regulation of anti-viral T cells following viral infection. Consequently, anti-viral T cell immunity was reduced in Qa-1b- and NKG2A-deficient mice, resulting in increased viral replication and immunopathology. NK cell depletion restored anti-viral immunity and virus control in the absence of Qa-1b. Taken together, our findings indicate that lymphocytes limit NK cell activity during viral infection in order to promote anti-viral T cell immunity.
PTP1B regulates Eph receptor function and trafficking
Nievergall, Eva; Janes, Peter W.; Stegmayer, Carolin; Vail, Mary E.; Haj, Fawaz G.; Teng, Shyh Wei; Neel, Benjamin G.; Bastiaens, Philippe I.; Lackmann, Martin
2010-01-01
Eph receptors orchestrate cell positioning during normal and oncogenic development. Their function is spatially and temporally controlled by protein tyrosine phosphatases (PTPs), but the underlying mechanisms are unclear and the identity of most regulatory PTPs are unknown. We demonstrate here that PTP1B governs signaling and biological activity of EphA3. Changes in PTP1B expression significantly affect duration and amplitude of EphA3 phosphorylation and biological function, whereas confocal ...
beta1 integrins are not required for the maintenance of lymphocytes within intestinal epithelia
DEFF Research Database (Denmark)
Marsal, Jan; Brakebusch, Cord; Bungartz, Gerd
2005-01-01
beta(1) integrins are thought to play a central role in maintaining lymphocytes within mucosal epithelia via their interactions with extracellular matrix proteins and subepithelial cellular components within and underlying the basement membrane. In the current study type a (CD8alphabetaTCRalphabe......beta(1) integrins are thought to play a central role in maintaining lymphocytes within mucosal epithelia via their interactions with extracellular matrix proteins and subepithelial cellular components within and underlying the basement membrane. In the current study type a (CD8alphabeta......TCRalphabeta) and type b (CD8alphaalphaTCRgammadelta and CD8alphaalphaTCRalphabeta) intraepithelial lymphocyte (IEL) subsets within the mouse small intestine were found to express functional beta(1) integrin and the beta(1) integrin alpha chain partners alpha(1), alpha(2), and alpha(4). Using inducible beta(1) integrin......-knockout bone marrow-chimeric mice we demonstrate that IEL expression of alpha(1) and alpha(2) but not alpha(4) is dependent on expression of the beta(1) chain. Importantly, deletion of the beta(1) chain in IEL did not alter the number or composition of lymphocytes within the intestinal epithelium. Thus, while...
Morinelli, Thomas A; Walker, Linda P; Velez, Juan Carlos Q; Ullian, Michael E
2015-02-05
The major effects of Angiotensin II (AngII) in vascular tissue are mediated by AngII AT1A receptor activation. Certain effects initiated by AT1A receptor activation require receptor internalization. In rat aortic vascular smooth muscle cells (RASMC), AngII stimulates cyclooxygenase 2 protein expression. We have previously shown this is mediated by β-arrestin-dependent receptor internalization and NF-κB activation. In this study, a specific inhibitor of clathrin-mediated endocytosis (CME), pitstop-2, was used to test the hypothesis that clathrin-dependent internalization of activated AT1A receptor mediates NF-κB activation and subsequent cyclooxygenase 2 expression. Radioligand binding assays, real time qt-PCR and immunoblotting were used to document the effects of pitstop-2 on AngII binding and signaling in RASMC. Laser scanning confocal microscopy (LSCM) was used to image pitstop-2׳s effects on AT1 receptor/GFP internalization in HEK-293 cells and p65 NF-κB nuclear localization in RASMC. Pitstop-2 significantly inhibited internalization of AT1A receptor (44.7% ± 3.1% Control vs. 13.2% ± 8.3% Pitstop-2; n=3) as determined by radioligand binding studies in RASMC. Studies utilizing AT1A receptor/GFP expressed in HEK 293 cells and LSCM confirmed these findings. Pitstop-2 significantly inhibited AngII-induced p65 NF-κB phosphorylation and nuclear localization, COX-2 message and protein expression in RASMC without altering activation of p42/44 ERK or TNFα signaling. Pitstop-2, a specific inhibitor of clathrin-mediated endocytosis, confirms that internalization of activated AT1A receptor mediates AngII activation of cyclooxygenase 2 expression in RASMC. These data provide support for additional intracellular signaling pathways activated through β-arrestin mediated internalization of G protein-coupled receptors, such as AT1A receptors. Copyright © 2014 Elsevier B.V. All rights reserved.
Salter, D M; Godolphin, J L; Gourlay, M S
1995-04-01
During development and at maturity different forms of cartilage vary in morphology and macromolecular content. This reflects heterogeneity of chondrocyte activity, in part involving differential interactions with the adjacent extracellular matrix via specialized cell surface receptors such as integrins. We undertook an immunohistological study on a series of human fetal knee joints to assess variation in the expression of integrins by chondrocytes and potential matrix ligands in articular, epiphyseal, growth plate, and meniscal cartilage. The results show that articular chondrocytes (beta 1+, beta 5 alpha V+, alpha 1+, alpha 2+/-, alpha 5+, weakly alpha 6+, alpha V+) differed from epiphyseal (beta 1+, beta 5 alpha V+, alpha 1+/-, alpha 2+/-, alpha 5+, alpha 6+, alpha V+) growth plate (beta 1+, beta 5 alpha V+, alpha 1-, alpha 2-, alpha 5+, alpha 6+, alpha V+), and meniscal cells (beta 1+, beta 5 alpha V+, alpha 1+, strongly alpha 2+, alpha 5+, alpha 6+, alpha V+ in expression of integrin subunits. There was no expression of beta 3, beta 4, beta 6, or alpha 3 by chondrocytes. These results differ from previous reports on the expression of integrins by adult articular cartilage, where alpha 2 and alpha 6 are not seen. Variation in distribution of matrix ligands was also seen. Fibronectin, laminin and Type VI collagen were expressed in all cartilages but there was restricted expression of tenascin, ED-A and ED-B fibronectin isoforms (articular cartilage and meniscus), and vitronectin (absent from growth plate cartilage). Regulated expression of integrins by chondrocytes, associated with changes in the pericellular matrix composition, is of potential importance in control of cartilage differentiation and function in health and disease.
DEFF Research Database (Denmark)
Mygind, Kasper J; Schwarz, Jeanette; Sahgal, Pranshu
2018-01-01
knockdown increases β1 integrin levels through mechanisms that are independent of its protease activity. In ADAM9-silenced cells, adhesion to collagen and fibronectin is reduced, suggesting an altered function of the accumulated integrins. Mechanistically, ADAM9 co-immunoprecipitates with β1 integrin......, and both internalization and subsequent degradation of β1 integrin are significantly decreased in ADAM9-silenced cells, with no effect on β1 integrin recycling. Accordingly, the formation of focal adhesions and actin stress fibres in ADAM9-silenced cells is altered, possibly explaining the reduction...
LENUS (Irish Health Repository)
McSherry, Elaine A
2011-03-23
Abstract Introduction The adhesion protein junctional adhesion molecule-A (JAM-A) regulates epithelial cell morphology and migration, and its over-expression has recently been linked with increased risk of metastasis in breast cancer patients. As cell migration is an early requirement for tumor metastasis, we sought to identify the JAM-A signalling events regulating migration in breast cancer cells. Methods MCF7 breast cancer cells (which express high endogenous levels of JAM-A) and primary cultures from breast cancer patients were used for this study. JAM-A was knocked down in MCF7 cells using siRNA to determine the consequences for cell adhesion, cell migration and the protein expression of various integrin subunits. As we had previously demonstrated a link between the expression of JAM-A and β1-integrin, we examined activation of the β1-integrin regulator Rap1 GTPase in response to JAM-A knockdown or functional antagonism. To test whether JAM-A, Rap1 and β1-integrin lie in a linear pathway, we tested functional inhibitors of all three proteins separately or together in migration assays. Finally we performed immunoprecipitations in MCF7 cells and primary breast cells to determine the binding partners connecting JAM-A to Rap1 activation. Results JAM-A knockdown in MCF7 breast cancer cells reduced adhesion to, and migration through, the β1-integrin substrate fibronectin. This was accompanied by reduced protein expression of β1-integrin and its binding partners αV- and α5-integrin. Rap1 activity was reduced in response to JAM-A knockdown or inhibition, and pharmacological inhibition of Rap1 reduced MCF7 cell migration. No additive anti-migratory effect was observed in response to simultaneous inhibition of JAM-A, Rap1 and β1-integrin, suggesting that they lie in a linear migratory pathway. Finally, in an attempt to elucidate the binding partners putatively linking JAM-A to Rap1 activation, we have demonstrated the formation of a complex between JAM-A, AF-6
LENUS (Irish Health Repository)
McSherry, Elaine A
2011-03-23
ABSTRACT: INTRODUCTION: The adhesion protein junctional adhesion molecule-A (JAM-A) regulates epithelial cell morphology and migration, and its over-expression has recently been linked with increased risk of metastasis in breast cancer patients. As cell migration is an early requirement for tumor metastasis, we sought to identify the JAM-A signalling events regulating migration in breast cancer cells. METHODS: MCF7 breast cancer cells (which express high endogenous levels of JAM-A) and primary cultures from breast cancer patients were used for this study. JAM-A was knocked down in MCF7 cells using siRNA to determine the consequences for cell adhesion, cell migration and the protein expression of various integrin subunits. As we had previously demonstrated a link between the expression of JAM-A and β1-integrin, we examined activation of the β1-integrin regulator Rap1 GTPase in response to JAM-A knockdown or functional antagonism. To test whether JAM-A, Rap1 and β1-integrin lie in a linear pathway, we tested functional inhibitors of all three proteins separately or together in migration assays. Finally we performed immunoprecipitations in MCF7 cells and primary breast cells to determine the binding partners connecting JAM-A to Rap1 activation. RESULTS: JAM-A knockdown in MCF7 breast cancer cells reduced adhesion to, and migration through, the β1-integrin substrate fibronectin. This was accompanied by reduced protein expression of β1-integrin and its binding partners αV- and α5-integrin. Rap1 activity was reduced in response to JAM-A knockdown or inhibition, and pharmacological inhibition of Rap1 reduced MCF7 cell migration. No additive anti-migratory effect was observed in response to simultaneous inhibition of JAM-A, Rap1 and β1-integrin, suggesting that they lie in a linear migratory pathway. Finally, in an attempt to elucidate the binding partners putatively linking JAM-A to Rap1 activation, we have demonstrated the formation of a complex between JAM-A, AF
LENUS (Irish Health Repository)
McSherry, Elaine A
2012-02-01
INTRODUCTION: The adhesion protein junctional adhesion molecule-A (JAM-A) regulates epithelial cell morphology and migration, and its over-expression has recently been linked with increased risk of metastasis in breast cancer patients. As cell migration is an early requirement for tumor metastasis, we sought to identify the JAM-A signalling events regulating migration in breast cancer cells. METHODS: MCF7 breast cancer cells (which express high endogenous levels of JAM-A) and primary cultures from breast cancer patients were used for this study. JAM-A was knocked down in MCF7 cells using siRNA to determine the consequences for cell adhesion, cell migration and the protein expression of various integrin subunits. As we had previously demonstrated a link between the expression of JAM-A and beta1-integrin, we examined activation of the beta1-integrin regulator Rap1 GTPase in response to JAM-A knockdown or functional antagonism. To test whether JAM-A, Rap1 and beta1-integrin lie in a linear pathway, we tested functional inhibitors of all three proteins separately or together in migration assays. Finally we performed immunoprecipitations in MCF7 cells and primary breast cells to determine the binding partners connecting JAM-A to Rap1 activation. RESULTS: JAM-A knockdown in MCF7 breast cancer cells reduced adhesion to, and migration through, the beta1-integrin substrate fibronectin. This was accompanied by reduced protein expression of beta1-integrin and its binding partners alphaV- and alpha5-integrin. Rap1 activity was reduced in response to JAM-A knockdown or inhibition, and pharmacological inhibition of Rap1 reduced MCF7 cell migration. No additive anti-migratory effect was observed in response to simultaneous inhibition of JAM-A, Rap1 and beta1-integrin, suggesting that they lie in a linear migratory pathway. Finally, in an attempt to elucidate the binding partners putatively linking JAM-A to Rap1 activation, we have demonstrated the formation of a complex between
Yong-Zheng, X; Wan-Li, M; Ji-Ming, M; Xue-Qun, R
2015-12-01
Nuclear factor-kB (NF-kB) activity is crucial for survival and proliferation of many kinds of malignancies, including gastric cancer (GC). The receptor for activated protein kinase C 1 (RACK1) is known to regulate tumor development, whereas the underlined mechanism has not been described clearly. We analyzed expression of RACK1 in paired human GC samples by both real-time polymerase chain reaction (PCR) and western blot. Effects of RACK inhibition with small interfering RNA or its overexpression in cultured GC cell lines were evaluated in cell viabilities. NF-kB signaling was investigated using luciferase reporter assay and real-time PCR. RACK1 was significantly decreased in GC samples. Knockdown of RACK elevated GC cell viabilities, whereas overexpression of RACK1 suppressed tumorigenesis of GC cells. Importantly, NF-kB signaling was enhanced after RACK1 expression was inhibited, suggesting the negative regulation of the pro-oncogenic NF-kB activity by RACK1 might contribute to its tumor suppressor role in GC cells. Our results support that RACK1 suppresses gastric tumor progression through the NF-kB signaling pathway.
Activation of GABAB receptors inhibits protein kinase B /Glycogen Synthase Kinase 3 signaling
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Lu Frances Fangjia
2012-11-01
Full Text Available Abstract Accumulated evidence has suggested that potentiation of cortical GABAergic inhibitory neurotransmission may be a key mechanism in the treatment of schizophrenia. However, the downstream molecular mechanisms related to GABA potentiation remain unexplored. Recent studies have suggested that dopamine D2 receptor antagonists, which are used in the clinical treatment of schizophrenia, modulate protein kinase B (Akt/glycogen synthase kinase (GSK-3 signaling. Here we report that activation of GABAB receptors significantly inhibits Akt/GSK-3 signaling in a β-arrestin-dependent pathway. Agonist stimulation of GABAB receptors enhances the phosphorylation of Akt (Thr-308 and enhances the phosphorylation of GSK-3α (Ser-21/β (Ser-9 in both HEK-293T cells expressing GABAB receptors and rat hippocampal slices. Furthermore, knocking down the expression of β-arrestin2 using siRNA abolishes the GABAB receptor-mediated modulation of GSK-3 signaling. Our data may help to identify potentially novel targets through which GABAB receptor agents may exert therapeutic effects in the treatment of schizophrenia.
Ferrer, Darío G; Dato, Virginia Actis; Fincati, Javier R Jaldín; Lorenc, Valeria E; Sánchez, María C; Chiabrando, Gustavo A
2017-07-01
Distinct modes of cell migration contribute to diverse types of cell movements. The mesenchymal mode is characterized by a multistep cycle of membrane protrusion, the formation of focal adhesion, and the stabilization at the leading edge associated with the degradation of extracellular matrix (ECM) components and with regulated extracellular proteolysis. Both α 2 -Macroglobulin (α 2 M) and its receptor, low density lipoprotein receptor-related protein 1 (LRP1), play important roles in inflammatory processes, by controlling the extracellular activity of several proteases. The binding of the active form of α 2 M (α 2 M*) to LRP1 can also activate different signaling pathways in macrophages, thus inducing extracellular matrix metalloproteinase-9 (MMP-9) activation and cellular proliferation. In the present study, we investigated whether the α 2 M*/LRP1 interaction induces cellular migration of the macrophage-derived cell line, Raw264.7. By using the wound-scratch migration assay and confocal microscopy, we demonstrate that α 2 M* induces LRP1-mediated mesenchymal cellular migration. This migration exhibits the production of enlarged cellular protrusions, MT1-MMP distribution to these leading edge protrusions, actin polymerization, focal adhesion formation, and increased intracellular LRP1/β1-integrin colocalization. Moreover, the presence of calphostin-C blocked the α 2 M*-stimulated cellular protrusions, suggesting that the PKC activation is involved in the cellular motility of Raw264.7 cells. These findings could constitute a therapeutic target for inflammatory processes with deleterious consequences for human health, such as rheumatoid arthritis, atherosclerosis and cancer. J. Cell. Biochem. 118: 1810-1818, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.
Ibrahim, Zein Shaban; Ahmed, Mohamed Mohamed; El-Shazly, Samir Ahmed; Ishizuka, Mayumi; Fujita, Shoichi
2014-01-01
Peroxisome proliferator activated receptor α (PPARα) ligands, fibrates used to control hyperlipidemia. We demonstrated CYP2B induction by clofibric acid (CFA) however, the mechanism was not clear. In this study, HepG2 cells transfected with expression plasmid of mouse constitutive androstane receptor (CAR) or PPARα were treated with CFA, phenobarbital (PB) or TCPOBOP. Luciferase assays showed that CFA increased CYP2B1 transcription to the same level as PB, or TCPOBOP in HepG2 transfected with mouse CAR But failed to induce it in PPARα transfected cells. CYP2B expressions were increased with PB or CFA in Wistar female rats (having normal levels of CAR) but not in Wistar Kyoto female rats (having low levels of CAR). The induction of CYP2B by PB or CFA was comparable to nuclear CAR levels. CAR nuclear translocation was induced by CFA in both rat strains. This indicates that fibrates can activate CAR and that fibrates-insulin sensitization effect may occur through CAR, while hypolipidemic effect may operate through PPARα.
Cross-talk between integrins α1β1 and α2β1 in renal epithelial cells
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Abair, Tristin D.; Sundaramoorthy, Munirathinam; Chen, Dong; Heino, Jyrki; Ivaska, Johanna; Hudson, Billy G.; Sanders, Charles R.; Pozzi, Ambra; Zent, Roy
2008-01-01
The collagen-binding integrins α1β1 and α2β1 have profoundly different functions, yet they are often co-expressed in epithelial cells. When both integrins are expressed in the same cell, it has been suggested that α1β1 negatively regulates integrin α2β1-dependent functions. In this study we utilized murine ureteric bud (UB) epithelial cells, which express no functionally detectable levels of endogenous integrins α1β1 and α2β1, to determine the mechanism whereby this regulation occurs. We demonstrate that UB cells expressing integrin α2β1, but not α1β1 adhere, migrate and proliferate on collagen I as well as form cellular cords in 3D collagen I gels. Substitution of the transmembrane domain of the integrin α2 subunit with that of α1 results in decreased cell adhesion, migration and cord formation. In contrast, substitution of the integrin α2 cytoplasmic tail with that of α1, decreases cell migration and cord formation, but increases proliferation. When integrin α1 and α2 subunits are co-expressed in UB cells, the α1 subunit negatively regulates integrin α2β1-dependent cord formation, adhesion and migration and this inhibition requires expression of both α1 and α2 tails. Thus, we provide evidence that the transmembrane and cytoplasmic domains of the α2 integrin subunit, as well as the α1 integrin subunit, regulate integrin α2β1 cell function
Salomone, Salvatore; Waeber, Christian
2011-01-01
Receptors for sphingosine-1-phosphate (S1P) have been identified only recently. Their medicinal chemistry is therefore still in its infancy, and few selective agonists or antagonists are available. Furthermore, the selectivity of S1P receptor agonists or antagonists is not well established. JTE-013 and BML-241 (also known as CAY10444), used extensively as specific S1P(2) and S1P(3) receptors antagonists respectively, are cases in point. When analyzing S1P-induced vasoconstriction in mouse basilar artery, we observed that JTE-013 inhibited not only the effect of S1P, but also the effect of U46619, endothelin-1 or high KCl; JTE-013 strongly inhibited responses to S1P in S1P(2) receptor knockout mice. Similarly, BML-241 has been shown to inhibit increases in intracellular Ca(2+) concentration via P(2) receptor or α(1A)-adrenoceptor stimulation and α(1A)-adrenoceptor-mediated contraction of rat mesenteric artery, while it did not affect S1P(3)-mediated decrease of forskolin-induced cyclic AMP accumulation. Another putative S1P(1/3) receptor antagonist, VPC23019, does not inhibit S1P(3)-mediated vasoconstriction. With these examples in mind, we discuss caveats about relying on available pharmacological tools to characterize receptor subtypes.
Directory of Open Access Journals (Sweden)
Christian eWaeber
2011-02-01
Full Text Available Receptors for sphingosine-1-phosphate (S1P have been identified only recently. Their medicinal chemistry is therefore still in its infancy, and few selective agonists or antagonists are available. Furthermore, the selectivity of S1P receptor agonists or antagonists is not well established. JTE-013 and BML-241 (also known as CAY10444, used extensively as specific S1P2 and S1P3 receptors antagonists respectively, are cases in point. When analyzing S1P-induced vasoconstriction in mouse basilar artery, we observed that JTE-013 inhibited not only the effect of S1P, but also the effect of U46619, endothelin-1 or high KCl; JTE-013 strongly inhibited responses to S1P in S1P2 receptor knockout mice. Similarly, BML-241 has been shown to inhibit increases in intracellular Ca2+ concentration via P2 receptor or α1A-adrenoceptor stimulation and α1A-adrenoceptor-mediated contraction of rat mesenteric artery, while it did not affect S1P3-mediated decrease of forskolin-induced cyclic AMP accumulation. Another putative S1P1/3 receptor antagonist, VPC23019, does not inhibit S1P3-mediated vasoconstriction. With these examples in mind, we discuss caveats about relying on available pharmacological tools to characterize receptor subtypes.
Leukocyte integrins and their ligand interactions
Hyun, Young-Min; Lefort, Craig T.; Kim, Minsoo
2010-01-01
Although critical for cell adhesion and migration during normal immune-mediated reactions, leukocyte integrins are also involved in the pathogenesis of diverse clinical conditions including autoimmune diseases and chronic inflammation. Leukocyte integrins therefore have been targets for anti-adhesive therapies to treat the inflammatory disorders. Recently, the therapeutic potential of integrin antagonists has been demonstrated in psoriasis and multiple sclerosis. However, current therapeutics broadly affect integrin functions and, thus, yield unfavorable side effects. This review discusses the major leukocyte integrins and the anti-adhesion strategies for treating immune diseases. PMID:19184539
Energy Technology Data Exchange (ETDEWEB)
O' Sullivan, Saoirse E [School of Biomedical Sciences, E Floor, Queen' s Medical Centre, University of Nottingham, Nottingham NG7 2UH (United Kingdom); Tarling, Elizabeth J [School of Biomedical Sciences, E Floor, Queen' s Medical Centre, University of Nottingham, Nottingham NG7 2UH (United Kingdom); Bennett, Andrew J [School of Biomedical Sciences, E Floor, Queen' s Medical Centre, University of Nottingham, Nottingham NG7 2UH (United Kingdom); Kendall, David A [School of Biomedical Sciences, E Floor, Queen' s Medical Centre, University of Nottingham, Nottingham NG7 2UH (United Kingdom); Randall, Michael D [School of Biomedical Sciences, E Floor, Queen' s Medical Centre, University of Nottingham, Nottingham NG7 2UH (United Kingdom)
2005-11-25
Cannabinoids have widespread effects on the cardiovascular system, only some of which are mediated via G-protein-coupled cell surface receptors. The active ingredient of cannabis, {delta}{sup 9}-tetrahydrocannabinol (THC), causes acute vasorelaxation in various arteries. Here we show for the first time that THC also causes slowly developing vasorelaxation through activation of peroxisome proliferator-activated receptors gamma (PPAR{gamma}). In vitro, THC (10 {mu}M) caused time-dependent vasorelaxation of rat isolated arteries. Time-dependent vasorelaxation to THC was similar to that produced by the PPAR{gamma} agonist rosiglitazone and was inhibited by the PPAR{gamma} antagonist GW9662 (1 {mu}M), but not the cannabinoid CB{sub 1} receptor antagonist AM251 (1 {mu}M). Time-dependent vasorelaxation to THC requires an intact endothelium, nitric oxide, production of hydrogen peroxide, and de novo protein synthesis. In transactivation assays in cultured HEK293 cells, THC-activated PPAR{gamma}, transiently expressed in combination with retinoid X receptor {alpha} and a luciferase reporter gene, in a concentration-dependent manner (100 nM-10 {mu}M). In vitro incubation with THC (1 or 10 {mu}M, 8 days) stimulated adipocyte differentiation in cultured 3T3L1 cells, a well-accepted property of PPAR{gamma} ligands. The present results provide strong evidence that THC is a PPAR{gamma} ligand, stimulation of which causes time-dependent vasorelaxation, implying some of the pleiotropic effects of cannabis may be mediated by nuclear receptors.
NMDA-induced potentiation of mGluR5 is mediated by activation of protein phosphatase 2B/calcineurin
Alagarsamy, Sudar; Saugstad, Julie; Warren, Lee; Mansuy, Isabelle M.; Gereau, Robert W.; Conn, P. Jeffrey
2010-01-01
Previous reports have shown that activation of N-methyl-D-aspartate (NMDA) receptors potentiates responses to activation of the group I metabotropic glutamate receptor mGluR5 by reversing PKC-mediated desensitization of this receptor. NMDA-induced reversal of mGluR5 desensitization is dependent on activation of protein phosphatases. However, the specific protein phosphatase involved and the precise mechanism by which NMDA receptor activation reduces mGluR desensitization are not known. We have performed a series of molecular, biochemical, and genetic studies to show that NMDA-induced regulation of mGluR5 is dependent on activation of calcium-dependent protein phosphatase 2B/calcineurin (PP2B/CaN). Furthermore, we report that purified calcineurin directly dephosphorylates the C-terminal tail of mGluR5 at sites that are phosphorylated by PKC. Finally, immunoprecipitation and GST fusion protein pull-down experiments reveal that calcineurin interacts with mGluR5, suggesting that these proteins could be colocalized in a signaling complex. Taken together with previous studies, these data suggest that activation of NMDA receptors leads to activation of calcineurin and that calcineurin modulates mGluR5 function by directly dephosphorylating mGluR5 at PKC sites that are involved in desensitization of this receptor. 2005 Elsevier Ltd. All rights reserved. PMID:16005030
Gatfield, John; Monnier, Lucile; Studer, Rolf; Bolli, Martin H; Steiner, Beat; Nayler, Oliver
2014-07-01
The sphingosine-1-phosphate (S1P) type 1 receptor (S1P1R) is a novel therapeutic target in lymphocyte-mediated autoimmune diseases. S1P1 receptor desensitization caused by synthetic S1P1 receptor agonists prevents T-lymphocyte egress from secondary lymphoid organs into the circulation. The selective S1P1 receptor agonist ponesimod, which is in development for the treatment of autoimmune diseases, efficiently reduces peripheral lymphocyte counts and displays efficacy in animal models of autoimmune disease. Using ponesimod and the natural ligand S1P, we investigated the molecular mechanisms leading to different signaling, desensitization and trafficking behavior of S1P1 receptors. In recombinant S1P1 receptor-expressing cells, ponesimod and S1P triggered Gαi protein-mediated signaling and β-arrestin recruitment with comparable potency and efficiency, but only ponesimod efficiently induced intracellular receptor accumulation. In human umbilical vein endothelial cells (HUVEC), ponesimod and S1P triggered translocation of the endogenous S1P1 receptor to the Golgi compartment. However, only ponesimod treatment caused efficient surface receptor depletion, receptor accumulation in the Golgi and degradation. Impedance measurements in HUVEC showed that ponesimod induced only short-lived Gαi protein-mediated signaling followed by resistance to further stimulation, whereas S1P induced sustained Gαi protein-mediated signaling without desensitization. Inhibition of S1P lyase activity in HUVEC rendered S1P an efficient S1P1 receptor internalizing compound and abrogated S1P-mediated sustained signaling. This suggests that S1P lyase - by facilitating S1P1 receptor recycling - is essential for S1P-mediated sustained signaling, and that synthetic agonists are functional antagonists because they are not S1P lyase substrates. Copyright © 2014 Elsevier Inc. All rights reserved.
DEFF Research Database (Denmark)
Brakebusch, Cord; Fillatreau, Simon; Potocnik, Alexandre J
2002-01-01
Several experimental evidences suggested that beta1 integrin-mediated adhesion of hematopoietic stem cells (HSC) is important for their function in the bone marrow (BM). Using induced deletion of the beta1 integrin gene restricted to the hematopoietic system, we show that beta1 integrin...... is not essential for HSC retention in the BM, hematopoiesis, and trafficking of lymphocytes. However, immunization with a T cell-dependent antigen resulted in virtually no IgM production and an increased secretion of IgG in mutant mice, while the response to a T cell-independent type 2 antigen showed decreases...
β1-adrenergic receptors activate two distinct signaling pathways in striatal neurons
Meitzen, John; Luoma, Jessie I.; Stern, Christopher M.; Mermelstein, Paul G.
2010-01-01
Monoamine action in the dorsal striatum and nucleus accumbens plays essential roles in striatal physiology. Although research often focuses on dopamine and its receptors, norepinephrine and adrenergic receptors are also crucial in regulating striatal function. While noradrenergic neurotransmission has been identified in the striatum, little is known regarding the signaling pathways activated by β-adrenergic receptors in this brain region. Using cultured striatal neurons, we characterized a novel signaling pathway by which activation of β1-adrenergic receptors leads to the rapid phosphorylation of cAMP Response Element Binding Protein (CREB), a transcription-factor implicated as a molecular switch underlying long-term changes in brain function. Norepinephrine-mediated CREB phosphorylation requires β1-adrenergic receptor stimulation of a receptor tyrosine kinase, ultimately leading to the activation of a Ras/Raf/MEK/MAPK/MSK signaling pathway. Activation of β1-adrenergic receptors also induces CRE-dependent transcription and increased c-fos expression. In addition, stimulation of β1-adrenergic receptors produces cAMP production, but surprisingly, β1-adrenergic receptor activation of adenylyl cyclase was not functionally linked to rapid CREB phosphorylation. These findings demonstrate that activation of β1-adrenergic receptors on striatal neurons can stimulate two distinct signaling pathways. These adrenergic actions can produce long-term changes in gene expression, as well as rapidly modulate cellular physiology. By elucidating the mechanisms by which norepinephrine and β1-adrenergic receptor activation affects striatal physiology, we provide the means to more fully understand the role of monoamines in modulating striatal function, specifically how norepinephrine and β1-adrenergic receptors may affect striatal physiology. PMID:21143600
International Nuclear Information System (INIS)
Sansing, Hope A.; Sarkeshik, Ali; Yates, John R.; Patel, Vyomesh; Gutkind, J. Silvio; Yamada, Kenneth M.; Berrier, Allison L.
2011-01-01
Research highlights: → Proteomics of clustered integrin αβ1, α v β, α 6 β receptors in oral carcinoma. → p130Cas, Dek, Src and talin regulate oral carcinoma invasion. → p130Cas, talin, Src and zyxin regulate oral carcinoma resistance to cisplatin. -- Abstract: Ligand engagement by integrins induces receptor clustering and formation of complexes at the integrin cytoplasmic face that controls cell signaling and cytoskeletal dynamics critical for adhesion-dependent processes. This study searches for a subset of integrin effectors that coordinates both tumor cell invasion and resistance to the chemotherapeutic drug cisplatin in oral carcinomas. Candidate integrin effectors were identified in a proteomics screen of proteins recruited to clustered integrin αβ1, α v β or α 6 β receptors in oral carcinomas. Proteins with diverse functions including microtubule and actin binding proteins, and factors involved in trafficking, transcription and translation were identified in oral carcinoma integrin complexes. Knockdown of effectors in the oral carcinoma HN12 cells revealed that p130Cas, Dek, Src and talin were required for invasion through Matrigel. Disruption of talin or p130Cas by RNA interference increased resistance to cisplatin, whereas targeting Dek, Src or zyxin reduced HN12 resistance to cisplatin. Analysis of the spreading of HN12 cells on collagen I and laminin I revealed that a decrease in p130Cas or talin expression inhibited spreading on both matrices. Interestingly, a reduction in zyxin expression enhanced spreading on laminin I and inhibited spreading on collagen I. Reduction of Dek, Src, talin or zyxin expression reduced HN12 proliferation by 30%. Proliferation was not affected by a reduction in p130Cas expression. We conclude that p130Cas, Src and talin function in both oral carcinoma invasion and resistance to cisplatin.
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Krause-Gruszczynska Malgorzata
2011-12-01
Full Text Available Abstract Background Host cell invasion by the foodborne pathogen Campylobacter jejuni is considered as one of the primary reasons of gut tissue damage, however, mechanisms and key factors involved in this process are widely unclear. It was reported that small Rho GTPases, including Cdc42, are activated and play a role during invasion, but the involved signaling cascades remained unknown. Here we utilised knockout cell lines derived from fibronectin-/-, integrin-beta1-/-, focal adhesion kinase (FAK-/- and Src/Yes/Fyn-/- deficient mice, and wild-type control cells, to investigate C. jejuni-induced mechanisms leading to Cdc42 activation and bacterial uptake. Results Using high-resolution scanning electron microscopy, GTPase pulldowns, G-Lisa and gentamicin protection assays we found that each studied host factor is necessary for induction of Cdc42-GTP and efficient invasion. Interestingly, filopodia formation and associated membrane dynamics linked to invasion were only seen during infection of wild-type but not in knockout cells. Infection of cells stably expressing integrin-beta1 variants with well-known defects in fibronectin fibril formation or FAK signaling also exhibited severe deficiencies in Cdc42 activation and bacterial invasion. We further demonstrated that infection of wild-type cells induces increasing amounts of phosphorylated FAK and growth factor receptors (EGFR and PDGFR during the course of infection, correlating with accumulating Cdc42-GTP levels and C. jejuni invasion over time. In studies using pharmacological inhibitors, silencing RNA (siRNA and dominant-negative expression constructs, EGFR, PDGFR and PI3-kinase appeared to represent other crucial components upstream of Cdc42 and invasion. siRNA and the use of Vav1/2-/- knockout cells further showed that the guanine exchange factor Vav2 is required for Cdc42 activation and maximal bacterial invasion. Overexpression of certain mutant constructs indicated that Vav2 is a linker
RPTP-alpha acts as a transducer of mechanical force on alphav/beta3-integrin-cytoskeleton linkages
DEFF Research Database (Denmark)
von Wichert, Gotz; Jiang, Guoying; Kostic, Ana
2003-01-01
Cell motility on ECM critically depends on the cellular response to force from the matrix. We find that force-dependent reinforcement of alphav/beta3-integrin-mediated cell-matrix connections requires the receptor-like tyrosine phosphatase alpha (RPTPalpha). RPTPalpha colocalizes with alphav...... of alphav/beta3-integrin-cytoskeleton connections during the initial phase of ECM contact. These observations indicate that Src family kinases have distinct functions during adhesion site assembly, and that RPTPalpha is an early component in force-dependent signal transduction pathways leading...
Integrin αv in the mechanical response of osteoblast lineage cells
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Kaneko, Keiko [Department of Bone and Joint Disease, National Center for Geriatrics and Gerontology, Obu, Aichi 474-8511 (Japan); Ito, Masako [Medical Work-Life-Balance Center, Nagasaki University Hospital, Nagasaki 852-8501 (Japan); Naoe, Yoshinori [Department of Mechanism of Aging, National Center for Geriatrics and Gerontology, Obu, Aichi 474-8511 (Japan); Lacy-Hulbert, Adam [Department of Pediatrics, Massachusetts General Hospital, Boston, MA 02114 (United States); Ikeda, Kyoji, E-mail: kikeda@ncgg.go.jp [Department of Bone and Joint Disease, National Center for Geriatrics and Gerontology, Obu, Aichi 474-8511 (Japan)
2014-05-02
Highlights: • Deletion of integrin αv in osteoblast lineage results in an impaired SOST response to loading in vivo. • c-Src–p130Cas–JNK–YAP/TAZ is activated via integrin αv on osteoblasts in response to FSS. • Deletion of integrin αv in osteoblasts results in impaired responses to mechanical stimulation. • Integrin αv is a key component of the mechanosensing machinery in bone. - Abstract: Although osteoblast lineage cells, especially osteocytes, are thought to be a primary mechanosensory cell in bone, the identity of the mechano-receptor and downstream mechano-signaling pathways remain largely unknown. Here we show using osteoblastic cell model of mechanical stimulation with fluid shear stress that in the absence of integrin αv, phosphorylation of the Src substrate p130Cas and JNK was impaired, culminating in an inhibition of nuclear translocation of YAP/TAZ and subsequent transcriptional activation of target genes. Targeted deletion of the integrin αv in osteoblast lineage cells results in an attenuated response to mechanical loading in terms of Sost gene expression, indicative of a role for integrin αv in mechanoreception in vivo. Thus, integrin αv may be integral to a mechanosensing machinery in osteoblastic cells and involved in activation of a Src–JNK–YAP/TAZ pathway in response to mechanical stimulation.
Integrin αv in the mechanical response of osteoblast lineage cells
International Nuclear Information System (INIS)
Kaneko, Keiko; Ito, Masako; Naoe, Yoshinori; Lacy-Hulbert, Adam; Ikeda, Kyoji
2014-01-01
Highlights: • Deletion of integrin αv in osteoblast lineage results in an impaired SOST response to loading in vivo. • c-Src–p130Cas–JNK–YAP/TAZ is activated via integrin αv on osteoblasts in response to FSS. • Deletion of integrin αv in osteoblasts results in impaired responses to mechanical stimulation. • Integrin αv is a key component of the mechanosensing machinery in bone. - Abstract: Although osteoblast lineage cells, especially osteocytes, are thought to be a primary mechanosensory cell in bone, the identity of the mechano-receptor and downstream mechano-signaling pathways remain largely unknown. Here we show using osteoblastic cell model of mechanical stimulation with fluid shear stress that in the absence of integrin αv, phosphorylation of the Src substrate p130Cas and JNK was impaired, culminating in an inhibition of nuclear translocation of YAP/TAZ and subsequent transcriptional activation of target genes. Targeted deletion of the integrin αv in osteoblast lineage cells results in an attenuated response to mechanical loading in terms of Sost gene expression, indicative of a role for integrin αv in mechanoreception in vivo. Thus, integrin αv may be integral to a mechanosensing machinery in osteoblastic cells and involved in activation of a Src–JNK–YAP/TAZ pathway in response to mechanical stimulation
Huang, Yu; Chen, Zhiying; Jang, Joon Hee; Baig, Mirza S; Bertolet, Grant; Schroeder, Casey; Huang, Shengjian; Hu, Qian; Zhao, Yong; Lewis, Dorothy E; Qin, Lidong; Zhu, Michael Xi; Liu, Dongfang
2018-04-18
The inhibitory receptor programmed cell death protein 1 (PD-1) is upregulated on a variety of immune cells, including natural killer (NK) cells, during chronic viral infection and tumorigenesis. Blockade of PD-1 or its ligands produces durable clinical responses with tolerable side effects in patients with a broad spectrum of cancers. However, the underlying molecular mechanisms of how PD-1 regulates NK cell function remain poorly characterized. We sought to determine the effect of PD-1 signaling on NK cells. PD-1 was overexpressed in CD16-KHYG-1 (a human NK cell line with both antibody-dependent cellular cytotoxicity through CD16 and natural cytotoxicity through NKG2D) cells and stimulated by exposing the cells to NK-sensitive target cells expressing programmed death ligand 1 (PD-L1). PD-1 engagement by PD-L1 specifically blocked NK cell-mediated cytotoxicity without interfering with the conjugation between NK cells and target cells. Further examination showed that PD-1 signaling blocked lytic granule polarization in NK cells, which was accompanied by failure of integrin-linked kinase, a key molecule in the integrin outside-in signaling pathway, to accumulate in the immunological synapse after NK-target cell conjugation. Our results suggest that NK cell cytotoxicity is inhibited by PD-1 engagement, which blocks lytic granule polarization to the NK cell immunological synapse with concomitant impairment of integrin outside-in signaling. This study provides novel mechanistic insights into how PD-1 inhibition disrupts NK cell function. Copyright © 2018 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.
Maniotis, A. J.; Chen, C. S.; Ingber, D. E.
1997-01-01
We report here that living cells and nuclei are hard-wired such that a mechanical tug on cell surface receptors can immediately change the organization of molecular assemblies in the cytoplasm and nucleus. When integrins were pulled by micromanipulating bound microbeads or micropipettes, cytoskeletal filaments reoriented, nuclei distorted, and nucleoli redistributed along the axis of the applied tension field. These effects were specific for integrins, independent of cortical membrane distortion, and were mediated by direct linkages between the cytoskeleton and nucleus. Actin microfilaments mediated force transfer to the nucleus at low strain; however, tearing of the actin gel resulted with greater distortion. In contrast, intermediate filaments effectively mediated force transfer to the nucleus under both conditions. These filament systems also acted as molecular guy wires to mechanically stiffen the nucleus and anchor it in place, whereas microtubules acted to hold open the intermediate filament lattice and to stabilize the nucleus against lateral compression. Molecular connections between integrins, cytoskeletal filaments, and nuclear scaffolds may therefore provide a discrete path for mechanical signal transfer through cells as well as a mechanism for producing integrated changes in cell and nuclear structure in response to changes in extracellular matrix adhesivity or mechanics.
Haanstra, Krista G.; Hofman, Sam O.; Estevao, Dave M. Lopes; Blezer, Erwin L. A.; Bauer, Jan; Yang, Li-Li; Wyant, Tim; Csizmadia, Vilmos; 't Hart, Bert A.; Fedyk, Eric R.
2013-01-01
The immune system is characterized by the preferential migration of lymphocytes through specific tissues (i.e., tissue tropism). Tissue tropism is mediated, in part, by the alpha(4) integrins expressed by T lymphocytes. The alpha(4)beta(1) integrin mediates migration of memory T lymphocytes into the
Cherian, Milu T; Lin, Wenwei; Wu, Jing; Chen, Taosheng
2015-05-01
Constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are xenobiotic sensors that enhance the detoxification and elimination of xenobiotics and endobiotics by modulating the expression of genes encoding drug-metabolizing enzymes and transporters. Elevated levels of drug-metabolizing enzymes and efflux transporters, resulting from CAR activation in various cancers, promote the elimination of chemotherapeutic agents, leading to reduced therapeutic effectiveness and acquired drug resistance. CAR inhibitors, in combination with existing chemotherapeutics, could therefore be used to attenuate multidrug resistance in cancers. Interestingly, all previously reported CAR inverse-agonists are also activators of PXR, rendering them mechanistically counterproductive in tissues where both these xenobiotic receptors are present and active. We used a directed high-throughput screening approach, followed by subsequent mechanistic studies, to identify novel, potent, and specific small-molecule CAR inhibitors that do not activate PXR. We describe here one such inhibitor, CINPA1 (CAR inhibitor not PXR activator 1), capable of reducing CAR-mediated transcription with an IC50 of ∼70 nM. CINPA1 1) is a specific xenobiotic receptor inhibitor and has no cytotoxic effects up to 30 µM; 2) inhibits CAR-mediated gene expression in primary human hepatocytes, where CAR is endogenously expressed; 3) does not alter the protein levels or subcellular localization of CAR; 4) increases corepressor and reduces coactivator interaction with the CAR ligand-binding domain in mammalian two-hybrid assays; and 5) disrupts CAR binding to the promoter regions of target genes in chromatin immunoprecipitation assays. CINPA1 could be used as a novel molecular tool for understanding CAR function. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.
Serotonin 2B Receptors in Mesoaccumbens Dopamine Pathway Regulate Cocaine Responses.
Doly, Stéphane; Quentin, Emily; Eddine, Raphaël; Tolu, Stefania; Fernandez, Sebastian P; Bertran-Gonzalez, Jesus; Valjent, Emmanuel; Belmer, Arnauld; Viñals, Xavier; Callebert, Jacques; Faure, Philippe; Meye, Frank J; Hervé, Denis; Robledo, Patricia; Mameli, Manuel; Launay, Jean-Marie; Maldonado, Rafael; Maroteaux, Luc
2017-10-25
Addiction is a maladaptive pattern of behavior following repeated use of reinforcing drugs in predisposed individuals, leading to lifelong changes. Common among these changes are alterations of neurons releasing dopamine in the ventral and dorsal territories of the striatum. The serotonin 5-HT 2B receptor has been involved in various behaviors, including impulsivity, response to antidepressants, and response to psychostimulants, pointing toward putative interactions with the dopamine system. Despite these findings, it remains unknown whether 5-HT 2B receptors directly modulate dopaminergic activity and the possible mechanisms involved. To answer these questions, we investigated the contribution of 5-HT 2B receptors to cocaine-dependent behavioral responses. Male mice permanently lacking 5-HT 2B receptors, even restricted to dopamine neurons, developed heightened cocaine-induced locomotor responses. Retrograde tracing combined with single-cell mRNA amplification indicated that 5-HT 2B receptors are expressed by mesolimbic dopamine neurons. In vivo and ex vivo electrophysiological recordings showed that 5-HT 2B -receptor inactivation in dopamine neurons affects their neuronal activity and increases AMPA-mediated over NMDA-mediated excitatory synaptic currents. These changes are associated with lower ventral striatum dopamine activity and blunted cocaine self-administration. These data identify the 5-HT 2B receptor as a pharmacological intermediate and provide mechanistic insight into attenuated dopamine tone following exposure to drugs of abuse. SIGNIFICANCE STATEMENT Here we report that mice lacking 5-HT 2B receptors totally or exclusively in dopamine neurons exhibit heightened cocaine-induced locomotor responses. Despite the sensitized state of these mice, we found that associated changes include lower ventral striatum dopamine activity and lower cocaine operant self-administration. We described the selective expression of 5-HT 2B receptors in a subpopulation of
The Receptor-Binding Domain in the VP1u Region of Parvovirus B19.
Leisi, Remo; Di Tommaso, Chiarina; Kempf, Christoph; Ros, Carlos
2016-02-24
Parvovirus B19 (B19V) is known as the human pathogen causing the mild childhood disease erythema infectiosum. B19V shows an extraordinary narrow tissue tropism for erythroid progenitor cells in the bone marrow, which is determined by a highly restricted uptake. We have previously shown that the specific internalization is mediated by the interaction of the viral protein 1 unique region (VP1u) with a yet unknown cellular receptor. To locate the receptor-binding domain (RBD) within the VP1u, we analyzed the effect of truncations and mutations on the internalization capacity of the recombinant protein into UT7/Epo cells. Here we report that the N-terminal amino acids 5-80 of the VP1u are necessary and sufficient for cellular binding and internalization; thus, this N-terminal region represents the RBD required for B19V uptake. Using site-directed mutagenesis, we further identified a cluster of important amino acids playing a critical role in VP1u internalization. In silico predictions and experimental results suggest that the RBD is structured as a rigid fold of three α-helices. Finally, we found that dimerization of the VP1u leads to a considerably enhanced cellular binding and internalization. Taken together, we identified the RBD that mediates B19V uptake and mapped functional and structural motifs within this sequence. The findings reveal insights into the uptake process of B19V, which contribute to understand the pathogenesis of the infection and the neutralization of the virus by the immune system.
Directory of Open Access Journals (Sweden)
Ky V. Hoang
2018-03-01
Full Text Available Francisella tularensis is a remarkably infectious facultative intracellular bacterium of macrophages that causes tularemia. Early evasion of host immune responses contributes to the success of F. tularensis as a pathogen. F. tularensis entry into human monocytes and macrophages is mediated by the major phagocytic receptor, complement receptor 3 (CR3, CD11b/CD18. We recently determined that despite a significant increase in macrophage uptake following C3 opsonization of the virulent Type A F. tularensis spp. tularensis Schu S4, this phagocytic pathway results in limited pro-inflammatory cytokine production. Notably, MAP kinase/ERK activation is suppressed immediately during C3-opsonized Schu S4-CR3 phagocytosis. A mathematical model of CR3-TLR2 crosstalk predicted early involvement of Ras GTPase-activating protein (RasGAP in immune suppression by CR3. Here, we link CR3-mediated uptake of opsonized Schu S4 by human monocytes and macrophages with inhibition of early signal 1 inflammasome activation, evidenced by limited caspase-1 cleavage and IL-18 release. This inhibition is due to increased RasGAP activity, leading to a reduction in the Ras-ERK signaling cascade upstream of the early inflammasome activation event. Thus, our data uncover a novel signaling pathway mediated by CR3 following engagement of opsonized virulent F. tularensis to limit inflammasome activation in human phagocytic cells, thereby contributing to evasion of the host innate immune system.
Accessibility of receptor-bound urokinase to type-1 plasminogen activator inhibitor
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Cubellis, M.V.; Andreasen, P.; Ragno, P.; Mayer, M.; Dano, K.; Blasi, F. (Univ. of Copenhagen (Denmark))
1989-07-01
Urokinase plasminogen activator (uPA) interacts with a surface receptor and with specific inhibitors, such as plasminogen activator inhibitor type 1 (PAI-1). These interactions are mediated by two functionally independent domains of the molecule: the catalytic domain (at the carboxyl terminus) and the growth factor domain (at the amino terminus). The authors have now investigated whether PAI-1 can bind and inhibit receptor-bound uPA. Binding of {sup 125}I-labeled ATF (amino-terminal fragment of uPA) to human U937 monocyte-like cells can be competed for by uPA-PAI-1 complexes, but not by PAI-1 alone. Preformed {sup 125}I-labeled uPA-PAI-1 complexes can bind to uPA receptor with the same binding specificity as uPA. PAI-1 also binds to, and inhibits the activity of, receptor-bound uPA in U937 cells, as shown in U937 cells by a caseinolytic plaque assay. Plasminogen activator activity of these cells is dependent on exogenous uPA, is competed for by receptor-binding diisopropyl fluorophosphate-treated uPA, and is inhibited by the addition of PAI-1. In conclusion, in U937 cells the binding to the receptor does not shield uPA from the action of PAI-1. The possibility that in adherent cells a different localization of PAI-1 and uPA leads to protection of uPA from PAI-1 is to be considered.
Capra, Valérie; Accomazzo, Maria Rosa; Gardoni, Fabrizio; Barbieri, Silvia; Rovati, G. Enrico
2010-01-01
Cysteinyl-leukotrienes (cysteinyl-LT) are rapidly generated at sites of inflammation and, in addition to their role in asthma, rhinitis, and other immune disorders, are increasingly regarded as significant inflammatory factors in cancer, gastrointestinal, cardiovascular diseases. We recently demonstrated that in monocyte/macrophage–like U937 cells, extracellular nucleotides heterologously desensitize CysLT1 receptor (CysLT1R)-induced Ca2+ transients. Given that monocytes express a number of other inflammatory and chemoattractant receptors, this study was aimed at characterizing transregulation between these different stimuli. We demonstrate that in U937 cells and in primary human monocytes, a series of inflammatory mediators activating Gi-coupled receptor (FPR1, BLT1) desensitize CysLT1R-induced Ca2+ response unidirectionally through activation of PKC. Conversely, PAF-R, exclusively coupled to Gq, cross-desensitizes CysLT1R without the apparent involvement of any kinase. Interestingly, Gs-coupled receptors (β2AR, H1/2R, EP2/4R) are also able to desensitize CysLT1R response through activation of PKA. Heterologous desensitization seems to affect mostly the Gi-mediated signaling of the CysLT1R. The hierarchy of desensitization among agonists may be important for leukocyte signal processing at the site of inflammation. Considering that monocytes/macrophages are likely to be the major source of cysteinyl-LT in many immunological and inflammatory processes, shedding light on how their receptors are regulated will certainly help to better understand the role of these cells in orchestrating this complex network of integrated signals. PMID:19965602
Transmission of integrin β7 transmembrane domain topology enables gut lymphoid tissue development.
Sun, Hao; Lagarrigue, Frederic; Gingras, Alexandre R; Fan, Zhichao; Ley, Klaus; Ginsberg, Mark H
2018-04-02
Integrin activation regulates adhesion, extracellular matrix assembly, and cell migration, thereby playing an indispensable role in development and in many pathological processes. A proline mutation in the central integrin β3 transmembrane domain (TMD) creates a flexible kink that uncouples the topology of the inner half of the TMD from the outer half. In this study, using leukocyte integrin α4β7, which enables development of gut-associated lymphoid tissue (GALT), we examined the biological effect of such a proline mutation and report that it impairs agonist-induced talin-mediated activation of integrin α4β7, thereby inhibiting rolling lymphocyte arrest, a key step in transmigration. Furthermore, the α4β7(L721P) mutation blocks lymphocyte homing to and development of the GALT. These studies show that impairing the ability of an integrin β TMD to transmit talin-induced TMD topology inhibits agonist-induced physiological integrin activation and biological function in development. © 2018 Sun et al.
Kim, Jae-Sung; Ellman, Michael B.; Yan, Dongyao; An, Howard S.; Kc, Ranjan; Li, Xin; Chen, Di; Xiao, Guozhi; Cs-Zabo, Gabriella; Hoskin, David W.; Buechter, D.D.; Van Wijnen, Andre J.; Im, Hee-Jeong
2013-01-01
The catabolic cytokine interleukin-1 (IL-1) and endotoxin lipopolysaccharide (LPS) are well-known inflammatory mediators involved in degenerative disc disease, and inhibitors of IL-1 and LPS may potentially be used to slow or prevent disc degeneration in vivo. Here, we elucidate the striking anti-catabolic and anti-inflammatory effects of bovine lactoferricin (LfcinB) in the intervertebral disc (IVD) via antagonism of both IL-1 and LPS-mediated catabolic activity using in vitro and ex vivo analyses. Specifically, we demonstrate the biological counteraction of LfcinB against IL-1 and LPS-mediated proteoglycan (PG) depletion, matrix-degrading enzyme production and enzyme activity in long-term (alginate beads) and short-term (monolayer) culture models using bovine and human nucleus pulposus (NP) cells. LfcinB significantly attenuates the IL-1 and LPS-mediated suppression of PG production and synthesis, and thus restores PG accumulation and pericellular matrix formation. Simultaneously, LfcinB antagonizes catabolic factor mediated induction of multiple cartilage-degrading enzymes, including MMP-1, MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5, in bovine NP cells at both mRNA and protein levels. LfcinB also suppresses the catabolic factor-induced stimulation of oxidative and inflammatory factors such as iNOS, IL-6, and toll-like receptor-2 (TLR-2) and TLR-4. Finally, the ability of LfcinB to antagonize IL-1 and LPS-mediated suppression of PG is upheld in an en bloc intradiscal microinjection model followed by ex vivo organ culture using both mouse and rabbit IVD tissue, suggesting a potential therapeutic benefit of LfcinB on degenerative disc disease in the future. PMID:23460134
Kim, Jae-Sung; Ellman, Michael B; Yan, Dongyao; An, Howard S; Kc, Ranjan; Li, Xin; Chen, Di; Xiao, Guozhi; Cs-Szabo, Gabriella; Hoskin, David W; Buechter, Doug D; Van Wijnen, Andre J; Im, Hee-Jeong
2013-09-01
The catabolic cytokine interleukin-1 (IL-1) and endotoxin lipopolysaccharide (LPS) are well-known inflammatory mediators involved in degenerative disc disease, and inhibitors of IL-1 and LPS may potentially be used to slow or prevent disc degeneration in vivo. Here, we elucidate the striking anti-catabolic and anti-inflammatory effects of bovine lactoferricin (LfcinB) in the intervertebral disc (IVD) via antagonism of both IL-1 and LPS-mediated catabolic activity using in vitro and ex vivo analyses. Specifically, we demonstrate the biological counteraction of LfcinB against IL-1 and LPS-mediated proteoglycan (PG) depletion, matrix-degrading enzyme production, and enzyme activity in long-term (alginate beads) and short-term (monolayer) culture models using bovine and human nucleus pulposus (NP) cells. LfcinB significantly attenuates the IL-1 and LPS-mediated suppression of PG production and synthesis, and thus restores PG accumulation and pericellular matrix formation. Simultaneously, LfcinB antagonizes catabolic factor mediated induction of multiple cartilage-degrading enzymes, including MMP-1, MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5, in bovine NP cells at both mRNA and protein levels. LfcinB also suppresses the catabolic factor-induced stimulation of oxidative and inflammatory factors such as iNOS, IL-6, and toll-like receptor-2 (TLR-2) and TLR-4. Finally, the ability of LfcinB to antagonize IL-1 and LPS-mediated suppression of PG is upheld in an en bloc intradiscal microinjection model followed by ex vivo organ culture using both mouse and rabbit IVD tissue, suggesting a potential therapeutic benefit of LfcinB on degenerative disc disease in the future. Copyright © 2013 Wiley Periodicals, Inc.
Gonzalez de Valdivia, Ernesto; Broselid, Stefan; Kahn, Robin; Olde, Björn; Leeb-Lundberg, L M Fredrik
2017-06-16
G protein-coupled receptor 30 (GPR30), also called G protein-coupled estrogen receptor 1 (GPER1), is thought to play important roles in breast cancer and cardiometabolic regulation, but many questions remain about ligand activation, effector coupling, and subcellular localization. We showed recently that GPR30 interacts through the C-terminal type I PDZ motif with SAP97 and protein kinase A (PKA)-anchoring protein (AKAP) 5, which anchor the receptor in the plasma membrane and mediate an apparently constitutive decrease in cAMP production independently of G i/o Here, we show that GPR30 also constitutively increases ERK1/2 activity. Removing the receptor PDZ motif or knocking down specifically AKAP5 inhibited the increase, showing that this increase also requires the PDZ interaction. However, the increase was inhibited by pertussis toxin as well as by wortmannin but not by AG1478, indicating that G i/o and phosphoinositide 3-kinase (PI3K) mediate the increase independently of epidermal growth factor receptor transactivation. FK506 and okadaic acid also inhibited the increase, implying that a protein phosphatase is involved. The proposed GPR30 agonist G-1 also increased ERK1/2 activity, but this increase was only observed at a level of receptor expression below that required for the constitutive increase. Furthermore, deleting the PDZ motif did not inhibit the G-1-stimulated increase. Based on these results, we propose that GPR30 increases ERK1/2 activity via two G i/o -mediated mechanisms, a PDZ-dependent, apparently constitutive mechanism and a PDZ-independent G-1-stimulated mechanism. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
The pathophysiological functions mediated by D-1 dopamine receptors
International Nuclear Information System (INIS)
Goldstein, M.; Kuga, S.; Meller, E.; SHimizu, Y.
1986-01-01
This chapter describes some behavioral responses which might be mediated by D 1 and D 2 DA receptors, and the authors discuss their clinical relevance. It was of considerable interest to determine whether a selective D 1 DA antagonist, such as SCH 23390, will induce catalepsy and whether this behavior is mediated by D 1 , or by both D 1 and D 2 DA receptors. Rats were used in the experiments. The authors examined whether the addition of the S 2 antagonist ketanserin affects the displacement of 3 H-Spi by SCH 23390. Induction of self-mutilating biting (SMB) behavior in monkeys with unilateral ventromedial tegmental (VMT) lesions by DA agonists and its prevention by DA antagonists is examined. The authors also discuss the possible relationships between abnormal guanine nucleotide metabolism and dopaminergic neuronal function through the implications in LeschNyhan syndrome and in some mental disorders
UNC5B receptor deletion exacerbates tissue injury in response to AKI.
Ranganathan, Punithavathi; Jayakumar, Calpurnia; Navankasattusas, Sutip; Li, Dean Y; Kim, Il-man; Ramesh, Ganesan
2014-02-01
Netrin-1 regulates cell survival and apoptosis by activation of its receptors, including UNC5B. However, the in vivo role of UNC5B in cell survival during cellular stress and tissue injury is unknown. We investigated the role of UNC5B in cell survival in response to stress using mice heterozygously expressing the UNC5B gene (UNC5B(-/flox)) and mice with targeted homozygous deletion of UNC5B in kidney epithelial cells (UNC5B(-/flox/GGT-cre)). Mice were subjected to two different models of organ injury: ischemia reperfusion injury of the kidney and cisplatin-induced nephrotoxicity. Both mouse models of UNC5B depletion had normal organ function and histology under basal conditions. After AKI, however, UNC5B(-/flox/GGT-cre) mice exhibited significantly worse renal function and damage, increased tubular apoptosis, enhanced p53 activation, and exacerbated inflammation compared with UNC5B(-/flox) and wild-type mice. shRNA-mediated suppression of UNC5B expression in cultured tubular epithelial cells exacerbated cisplatin-induced cell death in a p53-dependent manner and blunted Akt phosphorylation. Inhibition of PI3 kinase similarly exacerbated cisplatin-induced apoptosis; in contrast, overexpression of UNC5B reduced cisplatin-induced apoptosis in these cells. Taken together, these results show that the netrin-1 receptor UNC5B plays a critical role in cell survival and kidney injury through Akt-mediated inactivation of p53 in response to stress.
Ma, Jingyi; Stan Leung, L
2017-10-01
Decreased GABA B receptor function is proposed to mediate some symptoms of schizophrenia. In this study, we tested the effect of CGP7930, a GABA B receptor positive allosteric modulator, on ketamine-induced psychosis-relevant behaviors and hippocampal electrical activity in behaving rats. Electrodes were bilaterally implanted into the hippocampus, and cannulae were placed into the lateral ventricles of Long-Evans rats. CGP7930 or vehicle was injected intraperitoneally (i.p.) or intracerebroventricularly (i.c.v.), alone or 15 min prior to ketamine (3 mg/kg, subcutaneous) injection. Paired click auditory evoked potentials in the hippocampus (AEP), prepulse inhibition (PPI), and locomotor activity were recorded before and after drug injection. CGP7930 at doses of 1 mg/kg (i.p.) prevented ketamine-induced deficit of PPI. CGP7930 (1 mg/kg i.p.) also prevented the decrease in gating of hippocampal AEP and the increase in hippocampal gamma (65-100 Hz) waves induced by ketamine. Unilateral i.c.v. infusion of CGP7930 (0.3 mM/1 μL) also prevented the decrease in gating of hippocampal AEP induced by ketamine. Ketamine-induced behavioral hyperlocomotion was suppressed by 5 mg/kg i.p. CGP7930. CGP7930 alone, without ketamine, did not significantly affect integrated PPI, locomotion, gating of hippocampal AEP, or gamma waves. CGP7930 (1 mg/kg i.p.) increased heterosynaptically mediated paired pulse depression in the hippocampus, a measure of GABA B receptor function in vivo. CGP7930 reduces the behavioral and electrophysiological disruptions induced by ketamine in animals, and the hippocampus may be one of the neural targets where CGP7930 exerts its actions.
Directory of Open Access Journals (Sweden)
Shiva Akbarzadeh
2008-03-01
Full Text Available The transmembrane receptor 'ROR2' resembles members of the receptor tyrosine kinase family of signalling receptors in sequence but its' signal transduction mechanisms remain enigmatic. This problem has particular importance because mutations in ROR2 are associated with two human skeletal dysmorphology syndromes, recessive Robinow Syndrome (RS and dominant acting Brachydactyly type B (BDB. Here we show, using a constitutive dimerisation approach, that ROR2 exhibits dimerisation-induced tyrosine kinase activity and the ROR2 C-terminal domain, which is deleted in BDB, is required for recruitment and activation of the non-receptor tyrosine kinase Src. Native ROR2 phosphorylation is induced by the ligand Wnt5a and is blocked by pharmacological inhibition of Src kinase activity. Eight sites of Src-mediated ROR2 phosphorylation have been identified by mass spectrometry. Activation via tyrosine phosphorylation of ROR2 receptor leads to its internalisation into Rab5 positive endosomes. These findings show that BDB mutant receptors are defective in kinase activation as a result of failure to recruit Src.
Endosulfan induces CYP2B6 and CYP3A4 by activating the pregnane X receptor
International Nuclear Information System (INIS)
Casabar, Richard C.T.; Das, Parikshit C.; DeKrey, Gregory K.; Gardiner, Catherine S.; Cao Yan; Rose, Randy L.; Wallace, Andrew D.
2010-01-01
Endosulfan is an organochlorine pesticide commonly used in agriculture. Endosulfan has affects on vertebrate xenobiotic metabolism pathways that may be mediated, in part, by its ability to activate the pregnane X receptor (PXR) and/or the constitutive androstane receptor (CAR) which can elevate expression of cytochrome P450 (CYP) enzymes. This study examined the dose-dependency and receptor specificity of CYP induction in vitro and in vivo. The HepG2 cell line was transiently transfected with CYP2B6- and CYP3A4-luciferase promoter reporter plasmids along with human PXR (hPXR) or hCAR expression vectors. In the presence of hPXR, endosulfan-alpha exposure caused significant induction of CYP2B6 (16-fold) and CYP3A4 (11-fold) promoter activities over control at 10 μM. The metabolite endosulfan sulfate also induced CYP2B6 (12-fold) and CYP3A4 (6-fold) promoter activities over control at 10 μM. In the presence of hCAR-3, endosulfan-alpha induced CYP2B6 (2-fold) promoter activity at 10 μM, but not at lower concentrations. These data indicate that endosulfan-alpha significantly activates hPXR strongly and hCAR weakly. Using western blot analysis of human hepatocytes, the lowest concentrations at which CYP2B6 and CYP3A4 protein levels were found to be significantly elevated by endosulfan-alpha were 1.0 μM and 10 μM, respectively. In mPXR-null/hPXR-transgenic mice, endosulfan-alpha exposure (2.5 mg/kg/day) caused a significant reduction of tribromoethanol-induced sleep times by approximately 50%, whereas no significant change in sleep times was observed in PXR-null mice. These data support the role of endosulfan-alpha as a strong activator of PXR and inducer of CYP2B6 and CYP3A4, which may impact metabolism of CYP2B6 or CYP3A4 substrates.
Endosulfan induces CYP2B6 and CYP3A4 by activating the pregnane X receptor.
Casabar, Richard C T; Das, Parikshit C; Dekrey, Gregory K; Gardiner, Catherine S; Cao, Yan; Rose, Randy L; Wallace, Andrew D
2010-06-15
Endosulfan is an organochlorine pesticide commonly used in agriculture. Endosulfan has affects on vertebrate xenobiotic metabolism pathways that may be mediated, in part, by its ability to activate the pregnane X receptor (PXR) and/or the constitutive androstane receptor (CAR) which can elevate expression of cytochrome P450 (CYP) enzymes. This study examined the dose-dependency and receptor specificity of CYP induction in vitro and in vivo. The HepG2 cell line was transiently transfected with CYP2B6- and CYP3A4-luciferase promoter reporter plasmids along with human PXR (hPXR) or hCAR expression vectors. In the presence of hPXR, endosulfan-alpha exposure caused significant induction of CYP2B6 (16-fold) and CYP3A4 (11-fold) promoter activities over control at 10 microM. The metabolite endosulfan sulfate also induced CYP2B6 (12-fold) and CYP3A4 (6-fold) promoter activities over control at 10 microM. In the presence of hCAR-3, endosulfan-alpha induced CYP2B6 (2-fold) promoter activity at 10 microM, but not at lower concentrations. These data indicate that endosulfan-alpha significantly activates hPXR strongly and hCAR weakly. Using western blot analysis of human hepatocytes, the lowest concentrations at which CYP2B6 and CYP3A4 protein levels were found to be significantly elevated by endosulfan-alpha were 1.0 microM and 10 microM, respectively. In mPXR-null/hPXR-transgenic mice, endosulfan-alpha exposure (2.5mg/kg/day) caused a significant reduction of tribromoethanol-induced sleep times by approximately 50%, whereas no significant change in sleep times was observed in PXR-null mice. These data support the role of endosulfan-alpha as a strong activator of PXR and inducer of CYP2B6 and CYP3A4, which may impact metabolism of CYP2B6 or CYP3A4 substrates. Copyright 2010 Elsevier Inc. All rights reserved.
Barbieri, M Alejandro; Kong, Chen; Chen, Pin-I; Horazdovsky, Bruce F; Stahl, Philip D
2003-08-22
Activated epidermal growth factor receptors (EGFRs) recruit intracellular proteins that mediate receptor signaling and endocytic trafficking. Rin1, a multifunctional protein, has been shown to regulate EGFR internalization (1). Here we show that EGF stimulation induces a specific, rapid, and transient membrane recruitment of Rin1 and that recruitment is dependent on the Src homology 2 (SH2) domain of Rin1. Immunoprecipitation of EGFR is accompanied by co-immunoprecipitation of Rin1 in a time- and ligand-dependent manner. Association of Rin1 and specifically the SH2 domain of Rin1 with the EGFR was dependent on tyrosine phosphorylation of the intracellular domain of the EGFR. The recruitment of Rin1, observed by light microscopy, indicated that although initially cytosolic, Rin1 was recruited to both plasma membrane and endosomes following EGF addition. Moreover, the expression of the SH2 domain of Rin1 substantially impaired the internalization of EGF without affecting internalization of transferrin. Finally, we found that Rin1 co-immunoprecipitated with a number of tyrosine kinase receptors but not with cargo endocytic receptors. These results indicate that Rin1 provides a link via its SH2 domain between activated tyrosine kinase receptors and the endocytic pathway through the recruitment and activation of Rab5a.
Directory of Open Access Journals (Sweden)
Xiaoli Yuan
Full Text Available Given that integrin β1 is an important component of the connection to maintain glomerular structural integrity, by binding with multiple extracellular matrix proteins and mediating intracellular signaling. Focal adhesion kinase (FAK is the most essential intracellular integrator in the integrin β1-FAK signalling pathway. Here, we investigated the changes of the two molecules and visualized the possible interaction between them under various hemodynamic conditions in podocytes. Mice kidney tissues were prepared using in vivo cryotechnique (IVCT and then were stained and observed using light microscopy, confocal laser scanning microscopy and immunoelectron microscopy. The expression of these molecules were examined by western blot. Under the normal condition, integrin β1 stained continually and evenly at the membrane, and FAK was located in the cytoplasm and nuclei of the podocytes. There were significant colocalized plaques of two molecules. But under acute hypertensive and cardiac arrest conditions, integrin β1 decreased and stained intermittently. Similarly, FAK decreased and appeared uneven. Additionally, FAK translocated to the nuclei of the podocytes. As a result, the colocalization of integrin β1 and FAK reduced obviously under these conditions. Western blot assay showed a consistent result with the immunostaining. Collectively, the abnormal redistribution and decreased expressions of integrin β1 and FAK are important molecular events in regulating the functions of podocytes under abnormal hemodynamic conditions. IVCT could offer considerable advantages for morphological analysis when researching renal diseases.
Yamaguchi, Naoya; Mizutani, Takeomi; Kawabata, Kazushige; Haga, Hisashi
2015-01-07
Collective cell migration plays a crucial role in several biological processes, such as embryonic development, wound healing, and cancer metastasis. Here, we focused on collectively migrating Madin-Darby Canine Kidney (MDCK) epithelial cells that follow a leader cell on a collagen gel to clarify the mechanism of collective cell migration. First, we removed a leader cell from the migrating collective with a micromanipulator. This then caused disruption of the cohesive migration of cells that followed in movement, called "follower" cells, which showed the importance of leader cells. Next, we observed localization of active Rac, integrin β1, and PI3K. These molecules were clearly localized in the leading edge of leader cells, but not in follower cells. Live cell imaging using active Rac and active PI3K indicators was performed to elucidate the relationship between Rac, integrin β1, and PI3K. Finally, we demonstrated that the inhibition of these molecules resulted in the disruption of collective migration. Our findings not only demonstrated the significance of a leader cell in collective cell migration, but also showed that Rac, integrin β1, and PI3K are upregulated in leader cells and drive collective cell migration.
DeBell, Karen E.; Stoica, Bogdan A.; Verí, Maria-Concetta; Di Baldassarre, Angela; Miscia, Sebastiano; Graham, Laurie J.; Rellahan, Barbara L.; Ishiai, Masamichi; Kurosaki, Tomohiro; Bonvini, Ezio
1999-01-01
B-cell receptor (BCR)-induced activation of phospholipase C-γ1 (PLCγ1) and PLCγ2 is crucial for B-cell function. While several signaling molecules have been implicated in PLCγ activation, the mechanism coupling PLCγ to the BCR remains undefined. The role of PLCγ1 SH2 and SH3 domains at different steps of BCR-induced PLCγ1 activation was examined by reconstitution in a PLCγ-negative B-cell line. PLCγ1 membrane translocation required a functional SH2 N-terminal [SH2(N)] domain, was decreased by mutation of the SH3 domain, but was unaffected by mutation of the SH2(C) domain. Tyrosine phosphorylation did not require the SH2(C) or SH3 domains but depended exclusively on a functional SH2(N) domain, which mediated the association of PLCγ1 with the adapter protein, BLNK. Forcing PLCγ1 to the membrane via a myristoylation signal did not bypass the SH2(N) domain requirement for phosphorylation, indicating that the phosphorylation mediated by this domain is not due to membrane anchoring alone. Mutation of the SH2(N) or the SH2(C) domain abrogated BCR-stimulated phosphoinositide hydrolysis and signaling events, while mutation of the SH3 domain partially decreased signaling. PLCγ1 SH domains, therefore, have interrelated but distinct roles in BCR-induced PLCγ1 activation. PMID:10523627
Energy Technology Data Exchange (ETDEWEB)
Silva, Jose Alberto Alves da
2009-07-01
Gyroxin is a serine protease enzyme from the South American rattlesnake (Crotalus durissus terrificus) venom; it is only partially characterized and has multiple activities. Gyroxin induces blood coagulation, blood pressure decrease and a neurotoxic behavior named barrel rotation. The mechanisms involved in this neurotoxic activity are not known. Whereas gyroxin is a member of enzymes with high potential to become a new drug with clinical applications such as thrombin, batroxobin, ancrod, tripsyn and kalicrein, it is important to find out how gyroxin works. The analysis on agarose gel electrophoresis and circular dichroism confirmed the molecules' integrity and purity. The gyroxin intravenous administration in mice proved its neurotoxicity (barrel rotation). In vivo studies employing intravital microscopy proved that gyroxin induces vasodilation with the participation of protease activated receptors (PARs), nitric oxide and Na+K+ATPase. The leukocytes' adherence and rolling counting indicated that gyroxin has no pro inflammatory activity. Gyroxin induced platelet aggregation, which was blocked by inhibitors of PAR1 and PAR4 receptors (SCH 79797 and tcY-NH{sub 2}, respectively). Finally, it was proved that the gyroxin temporarily alter the permeability of the blood brain barrier (BBB). Our study has shown that both the protease-activated receptors and nitric oxide are mediators involved in the biological activities of gyroxin. (author)
Errasti, A E; Rey-Ares, V; Daray, F M; Rogines-Velo, M P; Sardi, S P; Paz, C; Podestá, E J; Rothlin, R P
2001-08-01
In isolated human umbilical vein (HUV), the contractile response to des-Arg9-bradykinin (des-Arg9-BK), selective BK B1 receptor agonist, increases as a function of the incubation time. Here, we evaluated whether cyclooxygenase (COX) pathway is involved in BK B1-sensitized response obtained in 5-h incubated HUV rings. The effect of different concentrations of indomethacin, sodium salicylate, ibuprofen, meloxicam, lysine clonixinate or NS-398 administrated 30 min before concentration-response curves (CRC) was studied. All treatments produced a significant rightward shift of the CRC to des-Arg9-BK in a concentration-dependent manner, which provides pharmacological evidence that COX pathway is involved in the BK B1 responses. Moreover, in this tissue, the NS-398 pKb (5.2) observed suggests that COX-2 pathway is the most relevant. The strong correlation between published pIC50 for COX-2 and the NSAIDs' pKbs estimated further supports the hypothesis that COX-2 metabolites are involved in BK B1 receptor-mediated responses. In other rings, indomethacin (30, 100 micromol/l) or NS-398 (10, 30 micromol/l) produced a significant rightward shift of the CRC to BK, selective BK B2 agonist, and its pKbs were similar to the values to inhibit BK B1 receptor responses, suggesting that COX-2 pathway also is involved in BK B2 receptor responses. Western blot analysis shows that COX-1 and COX-2 isoenzymes are present before and after 5-h in vitro incubation and apparently COX-2 does not suffer additional induction.
Zhang, Kai; Ding, Wei; Sun, Wei; Sun, Xiao-jiang; Xie, You-zhuan; Zhao, Chang-qing; Zhao, Jie
2016-01-01
Low back pain is associated with intervertebral disc degeneration (IVDD) due to cellular loss through apoptosis. Mechanical factors play an important role in maintaining the survival of the annulus fibrosus (AF) cells and the deposition of extracellular matrix. However, the mechanisms that excessive mechanical forces lead to AF cell apoptosis are not clear. The present study was to look for how AF cells sense mechanical changes. In vivo experiments, the involvement of mechanoreceptors in apoptosis was examined by RT-PCR and/or immunoblotting in the lumbar spine of rats subjected to unbalanced dynamic and static forces. In vitro experiments, we investigated apoptotic signaling pathways in untransfected and transfected AF cells with the lentivirus vector for rat β1 integrin overexpression after cyclic stretch. Apoptosis in AF cells was assessed using flow cytometry, Hoechst 33258 nuclear staining. Western blotting was used to analyze expression of β1 integrin and caspase-3 and ERK1/2 MAPK signaling molecules. In the rat IVDD model, unbalanced dynamic and static forces induced apoptosis of disc cells, which corresponded to decreased expression of β1 integrin. Cyclic stretch-induced apoptosis in rat AF cells correlated with the activation of caspase-3 and with decreased levels of β1 integrin and the phosphorylation levels of ERK1/2 activation level. However, the overexpression of β1 integrin in AF cells ameliorated cyclic stretch-induced apoptosis and decreased caspase-3 activation. Furthermore, ERK1/2-specific inhibitor promotes apoptosis in vector β1-infected AF cells. These results suggest that the disruption of β1 integrin signaling may underlie disc cell apoptosis induced by mechanical stress. Further work is necessary to fully elucidate the pathophysiological mechanisms that underlie IVDD caused by unbalanced dynamic and static forces.
Directory of Open Access Journals (Sweden)
Raffaella Iannone
2013-12-01
Full Text Available The A2b receptor (A2bR belongs to the adenosine receptor family. Emerging evidence suggest that A2bR is implicated in tumor progression in some murine tumor models, but the therapeutic potential of targeting A2bR in melanoma has not been examined. This study first shows that melanoma-bearing mice treated with Bay 60-6583, a selective A2bR agonist, had increased melanoma growth. This effect was associated with higher levels of immune regulatory mediators interleukin-10 (IL-10 and monocyte chemoattractant protein 1 (MCP-1 and accumulation of tumor-associated CD11b positive Gr1 positive cells (CD11b+Gr1+ myeloid-derived suppressor cells (MDSCs. Depletion of CD11b+Gr1+ cells completely reversed the protumor activity of Bay 60-6583. Conversely, pharmacological blockade of A2bR with PSB1115 reversed immune suppression in the tumor microenvironment, leading to a significant melanoma growth delay. PSB1115 treatment reduced both levels of IL-10 and MCP-1 and CD11b+Gr1+ cell number in melanoma lesions. These effects were associated with higher frequency of tumor-infiltrating CD8 positive (CD8+ T cells and natural killer T (NKT cells and increased levels of T helper 1 (Th1-like cytokines. Adoptive transfer of CD11b+Gr1+ cells abrogated the antitumor activity of PSB1115. These data suggest that the antitumor activity of PSB1115 relies on its ability to lower accumulation of tumor-infiltrating MDSCs and restore an efficient antitumor T cell response. The antitumor effect of PSB1115 was not observed in melanoma-bearing nude mice. Furthermore, PSB1115 enhanced the antitumor efficacy of dacarbazine. These data indicate that A2bR antagonists such as PSB1115 should be investigated as adjuvants in the treatment of melanoma.
Kousteni, Stavroula; Almeida, Maria; Han, Li; Bellido, Teresita; Jilka, Robert L; Manolagas, Stavros C
2007-02-01
Estrogens control gene transcription by cis or trans interactions of the estrogen receptor (ER) with target DNA or via the activation of cytoplasmic kinases. We report that selective activation of kinase-mediated actions of the ER with 4-estren-3alpha,17beta-diol (estren) or an estradiol-dendrimer conjugate, each a synthetic compound that stimulates kinase-mediated ER actions 1,000 to 10,000 times more potently than direct DNA interactions, induced osteoblastic differentiation in established cell lines of uncommitted osteoblast precursors and primary cultures of osteoblast progenitors by stimulating Wnt and BMP-2 signaling in a kinase-dependent manner. In sharp contrast, 17beta-estradiol (E(2)) suppressed BMP-2-induced osteoblast progenitor commitment and differentiation. Consistent with the in vitro findings, estren, but not E(2), stimulated Wnt/beta-catenin-mediated transcription in T-cell factor-lacZ transgenic mice. Moreover, E(2) stimulated BMP signaling in mice in which ERalpha lacks DNA binding activity and classical estrogen response element-mediated transcription (ERalpha(NERKI/-)) but not in wild-type controls. This evidence reveals for the first time the existence of a large signalosome in which inputs from the ER, kinases, bone morphogenetic proteins, and Wnt signaling converge to induce differentiation of osteoblast precursors. ER can either induce it or repress it, depending on whether the activating ligand (and presumably the resulting conformation of the receptor protein) precludes or accommodates ERE-mediated transcription.
International Nuclear Information System (INIS)
Aggarwal, Anshu; Al-Rohil, Rami N; Batra, Anupam; Feustel, Paul J; Jones, David M; DiPersio, C Michael
2014-01-01
Expression of integrin α3β1 is associated with tumor progression, metastasis, and poor prognosis in several cancers, including breast cancer. Moreover, preclinical studies have revealed important pro-tumorigenic and pro-metastatic functions for this integrin, including tumor growth, survival, invasion, and paracrine induction of angiogenesis. Our previously published work in a preclinical breast cancer model showed that integrin α3β1 promotes expression of cyclooxygenase-2 (COX2/PTGS2), a known driver of breast cancer progression. However, the clinical significance of this regulation was unknown. The objective of the current study was to assess the clinical relevance of the relationship between integrin α3β1 and COX2 by testing for their correlated expression among various forms of human breast cancer. Immunohistochemistry was performed to assess co-expression of α3 and COX2 in specimens of human invasive ductal carcinoma (IDC), either on a commercial tissue microarray (n = 59 samples) or obtained from Albany Medical Center archives (n = 68 samples). Immunostaining intensity for the integrin α3 subunit or COX2 was scored, and Spearman’s rank correlation coefficient analysis was performed to assess their co-expression across and within different tumor subtypes or clinicopathologic criteria. Although expression of integrin α3 or COX2 varied among clinical IDC samples, a statistically significant, positive correlation was detected between α3 and COX2 in both tissue microarrays (r s = 0.49, p < 0.001, n = 59) and archived samples (r s = 0.59, p < 0.0001, n = 68). In both sample sets, this correlation was independent of hormone receptor status, histological grade, or disease stage. COX2 and α3 are correlated in IDC independently of hormone receptor status or other clinicopathologic features, supporting the hypothesis that integrin α3β1 is a determinant of COX2 expression in human breast cancer. These results support the clinical relevance of α3β1
Cherian, Milu T.; Yang, Lei; Chai, Sergio C.; Lin, Wenwei; Chen, Taosheng
2016-01-01
The constitutive androstane receptor (CAR) regulates the expression of genes involved in drug metabolism and other processes. A specific inhibitor of CAR is critical for modulating constitutive CAR activity. We recently described a specific small-molecule inhibitor of CAR, CINPA1 (ethyl (5-(diethylglycyl)-10,11-dihydro-5H-dibenzo[b,f]azepin-3-yl)carbamate), which is capable of reducing CAR-mediated transcription by changing the coregulator recruitment pattern and reducing CAR occupancy at the...
Chen, Chiung-Nien; Chang, Cheng-Chi; Lai, Hong-Shiee; Jeng, Yung-Ming; Chen, Chia-I; Chang, King-Jeng; Lee, Po-Huang; Lee, Hsinyu
2015-07-01
Connective tissue growth factor (CTGF) plays important roles in normal and pathological conditions. The aim of this study was to investigate the role of CTGF in peritoneal metastasis as well as the underlying mechanism in gastric cancer progression. CTGF expression levels for wild-type and stable overexpression clones were determined by Western blotting and quantitative polymerase chain reaction (Q-PCR). Univariate and multivariate analyses, immunohistochemistry, and survival probability analyses were performed on gastric cancer patients. The extracellular matrix components involved in CTGF-regulated adhesion were determined. Recombinant CTGF was added to cells or coinoculated with gastric cancer cells into mice to evaluate its therapeutic potential. CTGF overexpression and treatment with the recombinant protein significantly inhibited cell adhesion. In vivo peritoneal metastasis demonstrated that CTGF-stable transfectants markedly decreased the number and size of tumor nodules in the mesentery. Statistical analysis of gastric cancer patient data showed that patients expressing higher CTGF levels had earlier TNM staging and a higher survival probability after the surgery. Integrin α3β1 was the cell adhesion molecule mediating gastric cancer cell adhesion to laminin, and blocking of integrin α3β1 prevented gastric cancer cell adhesion to recombinant CTGF. Coimmunoprecipitation results indicated that CTGF binds to integrin α3. Coinoculation of recombinant CTGF and gastric cancer cell lines in mice showed effective inhibition of peritoneal dissemination. Our results suggested that gastric cancer peritoneal metastasis is mediated through integrin α3β1 binding to laminin, and CTGF effectively blocks the interaction by binding to integrin α3β1, thus demonstrating the therapeutic potential of recombinant CTGF in gastric cancer patients.
Feedback regulation of mitochondria by caspase-9 in the B cell receptor-mediated apoptosis.
Eeva, J; Nuutinen, U; Ropponen, A; Mättö, M; Eray, M; Pellinen, R; Wahlfors, J; Pelkonen, J
2009-12-01
During the germinal centre reaction (GC), B cells with non-functional or self-reactive antigen receptors are negatively selected by apoptosis to generate B cell repertoire with appropriate antigen specificities. We studied the molecular mechanism of Fas/CD95- and B cell receptor (BCR)-induced apoptosis to shed light on the signalling events involved in the negative selection of GC B cells. As an experimental model, we used human follicular lymphoma (FL) cell line HF1A3, which originates from a GC B cell, and transfected HF1A3 cell lines overexpressing Bcl-x(L), c-FLIP(long) or dominant negative (DN) caspase-9. Fas-induced apoptosis was dependent on the caspase-8 activation, since the overexpression of c-FLIP(long), a natural inhibitor of caspase-8 activation, blocked apoptosis induced by Fas. In contrast, caspase-9 activation was not involved in Fas-induced apoptosis. BCR-induced apoptosis showed the typical characteristics of mitochondria-dependent (intrinsic) apoptosis. Firstly, the activation of caspase-9 was involved in BCR-induced DNA fragmentation, while caspase-8 showed only marginal role. Secondly, overexpression of Bcl-x(L) could block all apoptotic changes induced by BCR. As a novel finding, we demonstrate that caspase-9 can enhance the cytochrome-c release and collapse of mitochondrial membrane potential (DeltaPsi(m)) during BCR-induced apoptosis. The requirement of different signalling pathways in apoptosis induced by BCR and Fas may be relevant, since Fas- and BCR-induced apoptosis can thus be regulated independently, and targeted to different subsets of GC B cells.
Hood, Katie Y; Mair, Kirsty M; Harvey, Adam P; Montezano, Augusto C; Touyz, Rhian M; MacLean, Margaret R
2017-07-01
Serotonin can induce human pulmonary artery smooth muscle cell (hPASMC) proliferation through reactive oxygen species (ROS), influencing the development of pulmonary arterial hypertension (PAH). We hypothesize that in PASMCs, serotonin induces oxidative stress through NADPH-oxidase-derived ROS generation and reduced Nrf-2 (nuclear factor [erythroid-derived 2]-like 2) antioxidant systems, promoting vascular injury. HPASMCs from controls and PAH patients, and PASMCs from Nox1 -/- mice, were stimulated with serotonin in the absence/presence of inhibitors of Src kinase, the 5-HT 1B receptor, and NADPH oxidase 1 (Nox1). Markers of fibrosis were also determined. The pathophysiological significance of our findings was examined in vivo in serotonin transporter overexpressing female mice, a model of pulmonary hypertension. We confirmed thatserotonin increased superoxide and hydrogen peroxide production in these cells. For the first time, we show that serotonin increased oxidized protein tyrosine phosphatases and hyperoxidized peroxiredoxin and decreased Nrf-2 and catalase activity in hPASMCs. ROS generation was exaggerated and dependent on cellular Src-related kinase, 5-HT 1B receptor, and the serotonin transporter in human pulmonary artery smooth muscle cells from PAH subjects. Proliferation and extracellular matrix remodeling were exaggerated in human pulmonary artery smooth muscle cells from PAH subjects and dependent on 5-HT 1B receptor signaling and Nox1, confirmed in PASMCs from Nox1 -/- mice. In serotonin transporter overexpressing mice, SB216641, a 5-HT 1B receptor antagonist, prevented development of pulmonary hypertension in a ROS-dependent manner. Serotonin can induce cellular Src-related kinase-regulated Nox1-induced ROS and Nrf-2 dysregulation, contributing to increased post-translational oxidative modification of proteins and activation of redox-sensitive signaling pathways in hPASMCs, associated with mitogenic responses. 5-HT 1B receptors contribute to
International Nuclear Information System (INIS)
Lopez D, F.A.; Pedraza L, M.; Murphy, C.A. de; Ferro F, G.; Hernandez H, E.
2007-01-01
Full text: Nuclear medicine imaging techniques are non-invasive and monitor the spatiotemporal distribution of molecular events. Radiolabeled RGD-peptides are currently investigated to target integrin receptors for in vivo tumor imaging. The α v β 3 integrin is a target structure involved in the angio genesis process which mediates the binding to extracellular matrix via different proteins such as vitronectin, fibronectin and von Willebrand factor. The aim of this research was to prepare [ 99m Tc]-Lys-D-Phe-RGD and to evaluate its pharmacokinetics in athymic mice with three different induced malignant tumors. Tumor uptake values of 99m Tc-Lys-D-Phe-RGD labeled via HYNIC and EDDA showed good ability to target α v β 3 integrin receptors in the three different kinds of tumors (breast, prostate and neuroendocrine). A high in vivo stability and favorable pharmacokinetic properties such as fast blood clearance, rapid renal excretion, low liver and muscle uptake and low intestinal excretion were observed. This study demonstrated that 99m Tc-EDDA/HYNIC-Lys-D-Phe-RGD is a specific and potential radiopharmaceutical to image α v β 3 integrin receptors in a variety of tumors. (Author)
Directory of Open Access Journals (Sweden)
Margaret Veselits
Full Text Available Casitas B-lineage lymphoma-b (Cbl-b is a ubiquitin ligase (E3 that modulates signaling by tagging molecules for degradation. It is a complex protein with multiple domains and binding partners that are not involved in ubiquitinating substrates. Herein, we demonstrate that Cbl-b, but not c-Cbl, is recruited to the clustered B cell antigen receptor (BCR and that Cbl-b is required for entry of endocytosed BCRs into late endosomes. The E3 activity of Cbl-b is not necessary for BCR endocytic trafficking. Rather, the ubiquitin associated (UBA domain is required. Furthermore, the Cbl-b UBA domain is sufficient to confer the receptor trafficking functions of Cbl-b on c-Cbl. Cbl-b is also required for entry of the Toll-like receptor 9 (TLR9 into late endosomes and for the in vitro activation of TLR9 by BCR-captured ligands. These data indicate that Cbl-b acts as a scaffolding molecule to coordinate the delivery of the BCR and TLR9 into subcellular compartments required for productively delivering BCR-captured ligands to TLR9.
Energy Technology Data Exchange (ETDEWEB)
Sansing, Hope A. [Department of Oral and Craniofacial Biology, Louisiana State University Health Sciences Center-New Orleans, School of Dentistry, New Orleans, LA (United States); Sarkeshik, Ali; Yates, John R. [Department of Chemical Physiology, Scripps Research Institute, La Jolla, CA (United States); Patel, Vyomesh; Gutkind, J. Silvio [Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD (United States); Yamada, Kenneth M. [Laboratory of Cell and Developmental Biology, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD (United States); Berrier, Allison L., E-mail: allison.berrier@gmail.com [Department of Oral and Craniofacial Biology, Louisiana State University Health Sciences Center-New Orleans, School of Dentistry, New Orleans, LA (United States)
2011-03-11
Research highlights: {yields} Proteomics of clustered integrin {alpha}{beta}1, {alpha}{sub v}{beta}, {alpha}{sub 6}{beta} receptors in oral carcinoma. {yields} p130Cas, Dek, Src and talin regulate oral carcinoma invasion. {yields} p130Cas, talin, Src and zyxin regulate oral carcinoma resistance to cisplatin. -- Abstract: Ligand engagement by integrins induces receptor clustering and formation of complexes at the integrin cytoplasmic face that controls cell signaling and cytoskeletal dynamics critical for adhesion-dependent processes. This study searches for a subset of integrin effectors that coordinates both tumor cell invasion and resistance to the chemotherapeutic drug cisplatin in oral carcinomas. Candidate integrin effectors were identified in a proteomics screen of proteins recruited to clustered integrin {alpha}{beta}1, {alpha}{sub v}{beta} or {alpha}{sub 6}{beta} receptors in oral carcinomas. Proteins with diverse functions including microtubule and actin binding proteins, and factors involved in trafficking, transcription and translation were identified in oral carcinoma integrin complexes. Knockdown of effectors in the oral carcinoma HN12 cells revealed that p130Cas, Dek, Src and talin were required for invasion through Matrigel. Disruption of talin or p130Cas by RNA interference increased resistance to cisplatin, whereas targeting Dek, Src or zyxin reduced HN12 resistance to cisplatin. Analysis of the spreading of HN12 cells on collagen I and laminin I revealed that a decrease in p130Cas or talin expression inhibited spreading on both matrices. Interestingly, a reduction in zyxin expression enhanced spreading on laminin I and inhibited spreading on collagen I. Reduction of Dek, Src, talin or zyxin expression reduced HN12 proliferation by 30%. Proliferation was not affected by a reduction in p130Cas expression. We conclude that p130Cas, Src and talin function in both oral carcinoma invasion and resistance to cisplatin.
Energy Technology Data Exchange (ETDEWEB)
Heiss, Anika; Ammer, Hermann [Institute of Pharmacology, Toxicology and Pharmacy Ludwig-Maximilians-University of Munich Koeniginstrasse 16 80539 Muenchen Federal Republic of Germany (Germany); Eisinger, Daniela A., E-mail: eisinger@pharmtox.vetmed.uni-muenchen.de [Institute of Pharmacology, Toxicology and Pharmacy Ludwig-Maximilians-University of Munich Koeniginstrasse 16 80539 Muenchen Federal Republic of Germany (Germany)
2009-07-15
{delta}-Opioid receptor (DOR) agonists possess cytoprotective properties, an effect associated with activation of the 'pro-survival' kinase Akt. Here we delineate the signal transduction pathway by which opioids induce Akt activation in neuroblastoma x glioma (NG108-15) hybrid cells. Exposure of the cells to both [D-Pen{sup 2,5}]enkephalin and etorphine resulted in a time- and dose-dependent increase in Akt activity, as measured by means of an activation-specific antibody recognizing phosphoserine-473. DOR-mediated Akt signaling is blocked by the opioid antagonist naloxone and involves inhibitory G{sub i/o} proteins, because pre-treatment with pertussis toxin, but not over-expression of the G{sub q/11} scavengers EBP50 and GRK2-K220R, prevented this effect. Further studies with Wortmannin and LY294002 revealed that phophoinositol-3-kinase (PI3K) plays a central role in opioid-induced Akt activation. Opioids stimulate Akt activity through transactivation of receptor tyrosine kinases (RTK), because pre-treatment of the cells with inhibitors for neurotrophin receptor tyrosine kinases (AG879) and the insulin-like growth factor receptor IGF-1 (AG1024), but not over-expression of the G{beta}{gamma} scavenger phosducin, abolished this effect. Activated Akt translocates to the nuclear membrane, where it promotes GSK3 phosphorylation and prevents caspase-3 cleavage, two key events mediating inhibition of cell apoptosis and enhancement of cell survival. Taken together, these results demonstrate that in NG108-15 hybrid cells DOR agonists possess cytoprotective properties mediated by activation of the RTK/PI3K/Akt signaling pathway.
International Nuclear Information System (INIS)
Heiss, Anika; Ammer, Hermann; Eisinger, Daniela A.
2009-01-01
δ-Opioid receptor (DOR) agonists possess cytoprotective properties, an effect associated with activation of the 'pro-survival' kinase Akt. Here we delineate the signal transduction pathway by which opioids induce Akt activation in neuroblastoma x glioma (NG108-15) hybrid cells. Exposure of the cells to both [D-Pen 2,5 ]enkephalin and etorphine resulted in a time- and dose-dependent increase in Akt activity, as measured by means of an activation-specific antibody recognizing phosphoserine-473. DOR-mediated Akt signaling is blocked by the opioid antagonist naloxone and involves inhibitory G i/o proteins, because pre-treatment with pertussis toxin, but not over-expression of the G q/11 scavengers EBP50 and GRK2-K220R, prevented this effect. Further studies with Wortmannin and LY294002 revealed that phophoinositol-3-kinase (PI3K) plays a central role in opioid-induced Akt activation. Opioids stimulate Akt activity through transactivation of receptor tyrosine kinases (RTK), because pre-treatment of the cells with inhibitors for neurotrophin receptor tyrosine kinases (AG879) and the insulin-like growth factor receptor IGF-1 (AG1024), but not over-expression of the Gβγ scavenger phosducin, abolished this effect. Activated Akt translocates to the nuclear membrane, where it promotes GSK3 phosphorylation and prevents caspase-3 cleavage, two key events mediating inhibition of cell apoptosis and enhancement of cell survival. Taken together, these results demonstrate that in NG108-15 hybrid cells DOR agonists possess cytoprotective properties mediated by activation of the RTK/PI3K/Akt signaling pathway.
Ali, Hydar
2016-12-01
Mast cells (MCs) are tissue resident immune cells that are best known for their roles in allergic and inflammatory diseases. In addition to the high affinity IgE receptor (FcεRI), MCs express numerous G protein coupled receptors (GPCRs), which are the most common targets of drug therapy. Neurokinin 1 receptor (NK-1R) is expressed on MCs and contributes to IgE and non-IgE-mediated responses in mice. Although NK-1R antagonists are highly effective in modulating experimental allergic and inflammatory responses in mice they lack efficacy in humans. This article reviews recent findings that demonstrate that while neuropeptides (NPs) activate murine MCs via NK-1R and Mas related G protein coupled receptor B2 (MrgprB2), they activate human MCs via Mas-related G protein coupled receptor X2 (MRGPRX2). Interestingly, conventional NK-1R antagonists have off-target activity against mouse MrgprB2 but not human MRGPRX2. These findings suggest that the failure to translate studies with NK-1R antagonists from in vivo mouse studies to the clinic likely reflects their lack of effect on human MRGPRX2. A unique feature of MRGPRX2 that distinguishes it from other GPCRs is that it is activated by a diverse group of ligands that include; neuropeptides, cysteine proteases, antimicrobial peptides and cationic proteins released from activated eosinophils. Thus, the development of small molecule MRGPRX2-specific antagonists or neutralizing antibodies may provide new targets for the treatment of MC-mediated allergic and inflammatory diseases.
DeSmet, Marsha L; Fleet, James C
2017-10-01
High vitamin D status is associated with reduced colon cancer risk but these studies ignore the diversity in the molecular etiology of colon cancer. RAS activating mutations are common in colon cancer and they activate pro-proliferative signaling pathways. We examined the impact of RAS activating mutations on 1,25 dihydroxyvitamin D (1,25(OH) 2 D)-mediated gene expression in cultured colon and intestinal cell lines. Transient transfection of Caco-2 cells with a constitutively active mutant K-RAS (G12 V) significantly reduced 1,25(OH) 2 D-induced activity of both a human 25-hydroxyvitamin D, 24 hydroxyase (CYP24A1) promoter-luciferase and an artificial 3X vitamin D response element (VDRE) promoter-luciferase reporter gene. Young Adult Mouse Colon (YAMC) and Rat Intestinal Epithelial (RIE) cell lines with stable expression of mutant H-RAS had suppressed 1,25(OH) 2 D-mediated induction of CYP24A1 mRNA. The RAS effects were associated with lower Vitamin D receptor (VDR) mRNA and protein levels in YAMC and RIE cells and they could be partially reversed by VDR overexpression. RAS-mediated suppression of VDR levels was not due to either reduced VDR mRNA stability or increased VDR gene methylation. However, chromatin accessibility to the VDR gene at the proximal promoter (-300bp), an enhancer region at -6kb, and an enhancer region located in exon 3 was significantly reduced in RAS transformed YAMC cells (YAMC-RAS). These data show that constitutively active RAS signaling suppresses 1,25(OH) 2 D-mediated gene transcription in colon epithelial cells by reducing VDR gene transcription but the mechanism for this suppression is not yet known. These data suggest that cancers with RAS-activating mutations may be less responsive to vitamin D mediated treatment or chemoprevention. Copyright © 2017 Elsevier Ltd. All rights reserved.
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Duchene, Johan; Chauhan, Sharmila D.; Lopez, Frederic; Pecher, Christiane; Esteve, Jean-Pierre; Girolami, Jean-Pierre; Bascands, Jean-Loup; Schanstra, Joost P.
2005-01-01
Recently, we have described a novel protein-protein interaction between the G-protein coupled bradykinin B2 receptor and tyrosine phosphatase SHP-2 via an immunoreceptor tyrosine-based inhibition motif (ITIM) sequence located in the C-terminal part of the B2 receptor and the Src homology (SH2) domains of SHP-2. Here we show that phospholipase C (PLC)γ1, another SH2 domain containing protein, can also interact with this ITIM sequence. Using surface plasmon resonance analysis, we observed that PLCγ1 interacted with a peptide containing the phosphorylated form of the bradykinin B2 receptor ITIM sequence. In CHO cells expressing the wild-type B2 receptor, bradykinin-induced transient recruitment and activation of PLCγ1. Interestingly, this interaction was only observed in quiescent and not in proliferating cells. Mutation of the key ITIM residue abolished this interaction with and activation of PLCγ1. Finally we also identified bradykinin-induced PLCγ1 recruitment and activation in primary culture renal mesangial cells
Zhu, Xiang-Yu; Liu, Ning; Liu, Wei; Song, Shao-Wei; Guo, Ke-Jian
2012-04-01
Integrin-linked kinase (ILK) is an ankyrin repeat-containing serine-threonine protein kinase that is involved in the regulation of integrin-mediated processes such as cancer cell proliferation, migration and invasion. In this study, we examined the effect of a lentivirus-mediated knockdown of ILK on the proliferation, migration and invasion of pancreatic cancer (Panc-1) cells. Immunohistochemical staining showed that ILK expression was enhanced in pancreatic cancer tissue. The silencing of ILK in human Panc-1 cells led to cell cycle arrest in the G0/G1 phase and delayed cell proliferation, in addition to down-regulating cell migration and invasion. The latter effects were mediated by up-regulating the expression of E-cadherin, a key protein in cell adhesion. These findings indicate that ILK may be a new diagnostic marker for pancreatic cancer and that silencing ILK could be a potentially useful therapeutic approach for treating pancreatic cancer.
Targeting the urokinase plasminogen activator receptor inhibits ovarian cancer metastasis.
Kenny, Hilary A; Leonhardt, Payton; Ladanyi, Andras; Yamada, S Diane; Montag, Anthony; Im, Hae Kyung; Jagadeeswaran, Sujatha; Shaw, David E; Mazar, Andrew P; Lengyel, Ernst
2011-02-01
To understand the functional and preclinical efficacy of targeting the urokinase plasminogen activator receptor (u-PAR) in ovarian cancer. Expression of u-PAR was studied in 162 epithelial ovarian cancers, including 77 pairs of corresponding primary and metastatic tumors. The effect of an antibody against u-PAR (ATN-658) on proliferation, adhesion, invasion, apoptosis, and migration was assessed in 3 (SKOV3ip1, HeyA8, and CaOV3) ovarian cancer cell lines. The impact of the u-PAR antibody on tumor weight, number, and survival was examined in corresponding ovarian cancer xenograft models and the mechanism by which ATN-658 blocks metastasis was explored. Only 8% of all ovarian tumors were negative for u-PAR expression. Treatment of SKOV3ip1, HeyA8, and CaOV3 ovarian cancer cell lines with the u-PAR antibody inhibited cell invasion, migration, and adhesion. In vivo, anti-u-PAR treatment reduced the number of tumors and tumor weight in CaOV3 and SKOV3ip1 xenografts and reduced tumor weight and increased survival in HeyA8 xenografts. Immunostaining of CaOV3 xenograft tumors and ovarian cancer cell lines showed an increase in active-caspase 3 and TUNEL staining. Treatment with u-PAR antibody inhibited α(5)-integrin and u-PAR colocalization on primary human omental extracellular matrix. Anti-u-PAR treatment also decreased the expression of urokinase, u-PAR, β(3)-integrin, and fibroblast growth factor receptor-1 both in vitro and in vivo. This study shows that an antibody against u-PAR reduces metastasis, induces apoptosis, and reduces the interaction between u-PAR and α(5)-integrin. This provides a rationale for targeting the u-PAR pathway in patients with ovarian cancer and for further testing of ATN-658 in this indication. ©2010 AACR.
International Nuclear Information System (INIS)
Cho, Christina; Horzempa, Carol; Jones, David; McKeown-Longo, Paula J.
2016-01-01
Fibronectin is a mechanically sensitive protein which is organized in the extracellular matrix as a network of interacting fibrils. The lung tumor stroma is enriched for fibronectin which is thought to contribute to metastasis and drug resistance. Fibronectin is an elastic, multi-modular protein made up of individually folded domains, some of which can stretch in response to increased mechanical tension. Very little is known about the relationship of fibronectin’s unfolded domains to lung cancer resistance to chemotherapy. In the present study, we evaluated the impact of unfolding the first Type III domain of fibronectin (FnIII-1c) on TNF-related apoptosis inducing ligand (TRAIL) resistance. NCI-H460 non-small cell lung cancer cells were treated with FnIII-1c then assessed for TRAIL-induced apoptosis. Subsequent analysis of FnIII-1c-mediated signaling pathways was also completed. Human non-small cell lung cancer tissue sections were assessed for the expression of vitronectin by immunohistochemistry. FnIII-1c inhibited TRAIL-induced activation of caspase 8 and subsequent apoptosis in NCI-H460 lung cancer cells. FnIII-1c treatment was associated with the activation of the phosphatidylinositol-3-kinase/alpha serine/threonine kinase (PI3K/Akt) pathway and the αvβ5 integrin receptor for vitronectin, both of which were required for TRAIL resistance. Immunohistochemical staining of sections from non-small cell lung cancers showed that vitronectin was localized around blood vessels and in the tumor-stroma interface. Unfolding of Type III domains within the fibronectin matrix may promote TRAIL resistance through the activation of a PI3K/Akt/αvβ5 signaling axis and point to a novel mechanism by which changes in secondary structure of fibronectin contribute to cancer cell resistance to apoptosis
Directory of Open Access Journals (Sweden)
Thuy Tran
Full Text Available Expression of the α2β1 integrin, a receptor for collagens and laminin, is altered during tumor progression. Recent studies have linked polymorphisms in the α2 integrin gene with oral, squamous cell carcinoma (SCC. To determine the α2β1 integrin's role in SCC progression, we crossed α2-null mice with K14-HPV16 transgenic animals. Pathological progression to invasive carcinoma was evaluated in HPV-positive, α2-null (HPV/KO and HPV-positive, wild-type (HPV/WT animals. α2β1 integrin expression stimulated progression from hyperplasia and papillomatosis to dysplasia with concomitant dermal mast cell infiltration. Moreover, lymph node metastasis was decreased by 31.3% in HPV/KO, compared to HPV/WT, animals. To evaluate the integrin-specific impact on the malignant epithelium versus the microenvironment, we developed primary tumor cell lines. Although transition from dysplasia to carcinoma was unaltered during spontaneous tumor development, isolated primary HPV/KO SCC cell lines demonstrated decreased migration and invasion, compared to HPV/WT cells. When HPV/WT and HPV/KO SCC cells were orthotopically injected into WT or KO hosts, tumor α2β1 integrin expression resulted in decreased tumor latency, regardless of host integrin status. HPV/WT SCC lines failed to demonstrate a proliferative advantage in vitro, however, the HPV/WT tumors demonstrated increased growth compared to HPV/KO SCC lines in vivo. Although contributions of the integrin to the microenvironment cannot be excluded, our studies indicate that α2β1 integrin expression by HPV-transformed keratinocytes modulates SCC growth and progression.
International Nuclear Information System (INIS)
Oiwa, Ako; Kakizawa, Tomoko; Miyamoto, Takahide; Yamashita, Koh; Jiang, Wei; Takeda, Teiji; Suzuki, Satoru; Hashizume, Kiyoshi
2007-01-01
Small heterodimer partner (SHP; NR0B2) is an orphan nuclear receptor and acts as a repressor for wide variety of nuclear hormone receptors. We demonstrated here that mouse SHP mRNA showed a circadian expression pattern in the liver. Transient transfection of the mSHP promoter demonstrated that CLOCK-BMAL1, core circadian clock components, bound to E-box (CACGTG), and stimulated the promoter activity by 4-fold. Liver receptor homologue-1 (LRH-1; NR5A2) stimulated the mSHP promoter, and CLOCK-BMAL1 synergistically enhanced the LRH-1-mediated transactivation. Interestingly, SHP did not affect the CLOCK-BMAL1-mediated promoter activity, but strongly repressed the synergistic activation of CLOCK-BMAL1 and LRH-1. Furthermore, in vitro pull-down assays revealed the existence of direct protein-protein interaction between LRH-1 and CLOCK. In summary, this study shows that CLOCK-BMAL1, LRH-1 and SHP coordinately regulate the mSHP gene to generate the circadian oscillation. The cyclic expression of mSHP may affect daily activity of other nuclear receptors and contribute to circadian liver functions
Dickey, Deborah M; Edmund, Aaron B; Otto, Neil M; Chaffee, Thomas S; Robinson, Jerid W; Potter, Lincoln R
2016-05-20
C-type natriuretic peptide activation of guanylyl cyclase B (GC-B), also known as natriuretic peptide receptor B or NPR2, stimulates long bone growth, and missense mutations in GC-B cause dwarfism. Four such mutants (L658F, Y708C, R776W, and G959A) bound (125)I-C-type natriuretic peptide on the surface of cells but failed to synthesize cGMP in membrane GC assays. Immunofluorescence microscopy also indicated that the mutant receptors were on the cell surface. All mutant proteins were dephosphorylated and incompletely glycosylated, but dephosphorylation did not explain the inactivation because the mutations inactivated a "constitutively phosphorylated" enzyme. Tunicamycin inhibition of glycosylation in the endoplasmic reticulum or mutation of the Asn-24 glycosylation site decreased GC activity, but neither inhibition of glycosylation in the Golgi by N-acetylglucosaminyltransferase I gene inactivation nor PNGase F deglycosylation of fully processed GC-B reduced GC activity. We conclude that endoplasmic reticulum-mediated glycosylation is required for the formation of an active catalytic, but not ligand-binding domain, and that mutations that inhibit this process cause dwarfism. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
Lym-1 Chimeric Antigen Receptor T Cells Exhibit Potent Anti-Tumor Effects against B-Cell Lymphoma
Directory of Open Access Journals (Sweden)
Long Zheng
2017-12-01
Full Text Available T cells expressing chimeric antigen receptors (CARs recognizing CD19 epitopes have produced remarkable anti-tumor effects in patients with B-cell malignancies. However, cancer cells lacking recognized epitopes can emerge, leading to relapse and death. Thus, CAR T cells targeting different epitopes on different antigens could improve immunotherapy. The Lym-1 antibody targets a conformational epitope of Human Leukocyte Antigen-antigen D Related (HLA-DR on the surface of human B-cell lymphomas. Lym-1 CAR T cells were thus generated for evaluation of cytotoxic activity towards lymphoma cells in vitro and in vivo. Human T cells from healthy donors were transduced to express a Lym-1 CAR, and assessed for epitope-driven function in culture and towards Raji xenografts in NOD-scidIL2Rgammanull (NSG mice. Lym-1 CAR T cells exhibited epitope-driven activation and lytic function against human B-cell lymphoma cell lines in culture and mediated complete regression of Raji/Luciferase-Green fluorescent protein (Raji/Luc-GFP in NSG mice with similar or better reactivity than CD19 CAR T cells. Lym-1 CAR transduction of T cells is a promising immunotherapy for patients with Lym-1 epitope positive B-cell malignancies.
Mediator-dependent Nuclear Receptor Functions
Chen, Wei; Roeder, Robert
2011-01-01
As gene-specific transcription factors, nuclear hormone receptors are broadly involved in many important biological processes. Their function on target genes requires the stepwise assembly of different coactivator complexes that facilitate chromatin remodeling and subsequent preinitiation complex (PIC) formation and function. Mediator has proved to be a crucial, and general, nuclear receptor-interacting coactivator, with demonstrated functions in transcription steps ranging from chromatin remodeling to subsequent PIC formation and function. Here we discuss (i) our current understanding of pathways that nuclear receptors and other interacting cofactors employ to recruit Mediator to target gene enhancers and promoters, including conditional requirements for the strong NR-Mediator interactions mediated by the NR AF2 domain and the MED1 LXXLLL motifs and (ii) mechanisms by which Mediator acts to transmit signals from enhancer-bound nuclear receptors to the general transcription machinery at core promoters to effect PIC formation and function. PMID:21854863